CN104662423A

CN104662423A - Oligomeric Abeta in the diagnosis, prognosis, and monitoring of alzheimer's disease

Info

Publication number: CN104662423A
Application number: CN201380021783.5A
Authority: CN
Inventors: 丹尼尔·基德; 约翰尼斯·鲁尔夫·施特雷费尔
Original assignee: Janssen Alzheimer Immunotherapy
Current assignee: Janssen Sciences Ireland UC
Priority date: 2012-03-13
Filing date: 2013-03-13
Publication date: 2015-05-27
Also published as: US20130273574A1; JP2015511014A; HK1206423A1; WO2013138512A1; EP2825884A1; CA2867338A1; EP2825884A4

Abstract

Provided are methods for diagnosis, prognosis and monitoring of Alzheimer's disease. The methods involve measuring the amounts of combined monomeric and oligomeric Abeta and amount of monomeric Abeta in samples obtained from a subject, and determining a ratio. The ratio can be used in diagnosing, prognosing, and/or monitoring Alzheimer's disease.

Description

Oligomer A β in the diagnosis of A Zihai Mo's disease, prognosis and monitoring

With the cross reference of related application

The application is non-transitory, and the interests of require on March 13rd, 2012 to submit to 61/610,390, in this case all objects with its entirety by reference to being incorporated to herein.

Background technology

A Zihai Mo's disease (AD) is progressive disease (Selkoe, the TINS 16:403 (1993) causing senile dementia; Hardy etc., WO 92/13069; Selkoe, J.Neuropathol.Exp.Neurol.53:438 (1994); Duff etc., Nature 373:476 (1995); Games etc., Nature 373:523 (1995)).In broad terms, this disease is divided into two classes: the delayed occurred at old (65+ year), and the Early onset before the senility, namely fully appeared between 35 to 60 years old.In the disease of this two type, pathology are identical, but in the case started at comparatively early age, abnormal conditions are tending towards more serious and extensively.AD loses for feature with amyloid plaques, NFT and brain neuron.NFT is the intracellular deposits of microtubule associativity Tau albumen, and it is formed by being mutually wound in two right filaments.Amyloid plaques is the unordered nerve fibre web area of span up to 150 μm, has extracellular amyloid sediment in center, and its microscopic analysis can cut into slices by cerebral tissue is seen.Early onset A Zihai Mo's disease is relevant to No. 21 chromosomal trisomies in the genetic mutation in especially APP or presenilin gene and Down syndrome.

The primary constituent of amyloid plaques is the peptide being called as A β.A β is processed by the proteolysis of large transmembrane glycoprotein, i.e. amyloid precusor protein (APP) and produces.The length of A β be 39 to 43 amino acid not etc.Dominant forms A β 40 length is 40 amino acid, and is considered to short-form.Second common form A β 42 length is 42 amino acid, and is considered to microscler formula.A β 42 is relevant to pathogenicity, and is the main constituent in neuritic plaques (90%) and essence blood vessel sediment (75%).Roher etc., Proc.Nat ' l Acad.Sci.USA90:10836 (1993).The c-terminus of A β comprises a part for the hydrophobic transmembrane domain of APP, and this can explain that it is gathered into the tendentiousness of the fibril forming patch.

The gradual brain deposition of A β can occur in cognitive symptom before several years or even many decades (Selkoe, J.Neuropath.and Exp.Neurol.53:438 (1994) and Selkoe, Neuron6:487 (1991)).The formation of amyloid plaques and/or other the physically different determination methods with disease association were detected before occurring in cognitive symptom, can so that the treatment of AD and prevention.Large biosy of brain tissue is very invasive and is therefore undesired, especially in the experimenter not showing cognitive symptom.Report, the in-vivo imaging of amyloid deposition is that brain is bioptic can alternative scheme (WO11/106732), but imaging technique needs the complicated and equipment of costliness, and needs professional to carry out interpretation of images.

Another kind method is the biomarker in test set tissue samples, especially body fluid.Reported compared with normal healthy controls, in the cerebrospinal fluid (CSF) of experimenter suffering from AD, the level of solubility A β 42 is lower.Report the another kind of biomarker Tau discharged by neuronal cell injury, in the CSF of AD patient, raised (Vandermeeren etc., J.Neurochem.61:1828 (1993)).Propose the measurement of solubility A β and/or soluble T au to be used for diagnosis and monitoring AD (see such as U.S. Patent number 7,700,309).But in non-ad and AD experimenter, the overlapping ranges of these biomarkers, causes false negative and false positive.

Summary of the invention

The invention provides the method for the diagnosis of auxiliary A Zihai Mo's disease or its neurological susceptibility, prognosis or monitoring.Such method comprises: (a) measures the amount coming from monomer A β in the humoral sample of experimenter; B () measures the amount coming from monomer and oligomer A β in second humoral sample of described experimenter; C the amount of the amount of described monomer A β and monomer and oligomer A β compares by (); And (d) compares described for the diagnosis of A Zihai Mo's disease or its neurological susceptibility in described experimenter, prognosis or monitoring.Some method determination monomer A β and the ratio between monomer and oligomer A β, monomer A β, than the less quotient of monomer and oligomer A β, indicates the deterioration of the higher neurological susceptibility of the described disease of generation of described experimenter, higher possibility that described disease exists or the state of an illness.Ratio between some method determination monomer A β and oligomer A β, monomer A β, than the less quotient of oligomer A β, indicates the deterioration of the higher neurological susceptibility of the described disease of generation of described experimenter, higher possibility that described disease exists or the state of an illness.The amount of some method determination oligomer A β, the higher amount of oligomer A β, indicates the deterioration of the higher neurological susceptibility of the described disease of generation of described experimenter, higher possibility that described disease exists or the state of an illness.

Some method measures at least one in A β x-37, A β x-38, A β x-39, A β x-40, A β x-41 and A β x-42.Some method at least measures A β x-40.Some method at least measures A β x-42.Some method at least measures A β x-40 and A β x-42.

In some method, the amount of monomer A β uses one or more antibody to measure, and described antibody holds epi-position in conjunction with one or more C-, and described C-holds epi-position exist in monomer A β and do not exist in oligomer A β.In some method, described one or more C-hold antibody to be one or more end-specific antibodies for A β 37, A β 38, A β 39, A β 40, A β 41 or A β 42.In some method, one or more C-described hold antibody to comprise the antibody of terminal specificity for A β 40, are optionally antibody 2G3.In some method, one or more C-described hold antibody to comprise the antibody of terminal specificity for A β 42, are optionally antibody 21F12.In some method, described one or more C-hold antibody to comprise terminal specificity for the antibody of A β 40 and the terminal specificity antibody for A β 42.In some method, described monomer A β is measured by the affine sandwich assay of immunity, and described determination method comprises one or more C-described and holds antibody and the another kind of antibody in conjunction with N-end and/or central epi-position.In some method, described another kind of antibody holds epi-position in conjunction with N-, is optionally antibody 3D6.In some method, described another kind of antibody, in conjunction with central epi-position, is optionally antibody 266.In some method, one or more C-described hold antibody to be reporter antibody, and described another kind of antibody is capture antibody.In some method, one or more C-described hold antibody to be capture antibody, and described another kind of antibody is reporter antibody.In some method, one or more reporter antibody rutheniums described mark, and described capture antibody biotin labeling.

In some method, measure the amount of monomer and oligomer A β in the step (b), comprise with sample described in depolymerization agent process oligomer A β being transformed into monomer A β, and measure the amount of monomer A β in the sample of described depolymerization agent process.In some method, described depolymerization agent comprises chaotropic agent so that oligomer is dissolved into monomer.Chaotropic agent comprises guanidine hydrochloride, guanidinium isothiocyanate, urea, thiocarbamide, lithium perchlorate and/or potassium iodide.In some method, described depolymerization agent comprises non-ionic detergent.In some method, described depolymerization agent comprises polyglycol, polyvinylpyrrolidone, polyphenol and/or some Small molecular such as hexafluoroisopropanol.In some method, the amount of monomer A β in the sample of described depolymerization agent process, by measuring for the determination method that the amount measuring monomer A β is identical with step (a).In some method, described measuring process is undertaken by quantitative mass spectral art.In some method, described measuring process, by kapillary or gel electrophoresis, is then undertaken by quantitative Western blot method.

In some method, described humoral sample is CSF sample.In some method, described humoral sample is blood sample.In some method, described blood sample is plasma sample.In some method, step (a) and (b) carry out simultaneously.In some method, the sample of described step (a) and the second sample of described step (b) are the different sample aliquot coming from simple sample.

In some method, described experimenter does not have cognitive impairment, and step (d) assesses the neurological susceptibility that A Zihai Mo's disease occurs described experimenter.In some method, described experimenter has mild cognitive impairment, and step (d) assesses the neurological susceptibility that A Zihai Mo's disease occurs described experimenter.In some method, described experimenter has cognitive impairment, and step (d) comprise use step (c) comparison and other symptoms of described experimenter's illness and/or the combination of sign to provide the diagnosis of A Zihai Mo's disease.In some method, described experimenter had been diagnosed as before carrying out described method suffers from A Zihai Mo's disease, and step (d) provides the instruction of described disease stage.In some method, described experimenter is just accepting treatment or the prevention of A Zihai Mo's disease, and step (d) provides described experimenter to the instruction of the response for the treatment of.In some method, described method is carried out at set intervals on same experimenter, and step (c) provide the instruction of response of described experimenter to treatment more over time.

In some method, described experimenter is just with the Immuno Suppressive Therapy for A β.In some method, described experimenter is just using hundred flat pearl monoclonal antibody (bapineuzumab) treatments.Before some method is also included in and carries out step (a) and (b), with the anti-idiotype for hundred flat pearl monoclonal antibodies, optionally with the described sample of JH11.22G2 process.Some method also comprises the amount measuring Tau or P-Tau in described sample, wherein Tau or P-Tau is relative to the increase of control value, provides the neurological susceptibility of generation A Zihai Mo's disease of described experimenter, the existence of A Zihai Mo's disease or the further instruction that sb.'s illness took a turn for the worse.

In some method, described experimenter enters clinical testing to test the candidate for the treatment of A Zihai Mo's disease or the medicine of prevention, if wherein described monomer A β than the quotient of monomer and oligomer A β lower than threshold value, then described experimenter is comprised in described clinical testing, if described experimenter is higher than described threshold value, then described experimenter is excluded from described clinical testing.Some method also comprises the nursing supplier described diagnosis, prognosis or monitoring being informed described experimenter or described experimenter.

In some method, at least described step comparing the amount of monomer A β and monomer and oligomer A β performs in a computer.In some method, described computing machine receives the signal relevant with the amount of monomer A β and the amount of monomer and oligomer A β, described signal is transformed into quantitative amount, more described amount quantitatively, and provide and treat relevant output with the recommendation of the comparison of described amount, described amount, the situation of described experimenter or described experimenter.

Present invention also offers and determine to which the snibject's medicine in colony with the method for the prevention or treatment of carrying out A Zihai Mo's disease.Such method comprises each experimenter in described colony: (a) measures the amount of monomer A β in humoral sample; B () measures the amount of monomer and oligomer A β in the second sample of body fluid; And the amount of the amount of monomer A β and monomer and oligomer A β compares by (c), wherein on basis of the comparison, experimenter in described colony accepts or does not accept medicine to treat A Zihai Mo's disease or to carry out the prevention of A Zihai Mo's disease.In some method, described comparison determines monomer A β and the ratio between monomer and oligomer A β, and wherein monomer A β accepts described medicine than the quotient of monomer and oligomer A β lower than the experimenter of threshold value.

Present invention also offers the method determining which kind of therapeutic scheme of experimenter given in colony.Such method needs the amount measuring monomer A β in humoral sample; Measure the amount of monomer and oligomer A β in the second sample of body fluid; And the amount of the amount of monomer A β and monomer and oligomer A β is compared.First sub-group of described experimenter is treated with the first therapeutic scheme, second sub-group of described experimenter is treated with the second therapeutic scheme, wherein between the experimenter and the experimenter of the second sub-group of described first sub-group, monomer A β is significantly more different than the ratio of monomer and oligomer A β.In the method that some is such, described first therapeutic scheme comprises the medicine of prevention for A Zihai Mo's disease or treatment, described second therapeutic scheme does not comprise described medicine, and the experimenter of described first sub-group has the ratio of lower monomer A β than monomer and oligomer A β compared with the experimenter of described second sub-group.In the method that some is such, described monomer A β than the quotient of monomer and oligomer A β in the experimenter of described first sub-group lower than threshold value, and lower than threshold value in the experimenter of described second sub-group.The measurement of A beta form and the calculating of the parameter relevant to oligomer A β can be carried out according to any method described herein.

Present invention also offers the method for the experimenter in colony being carried out to difference treatment, described method comprises first sub-group for the treatment of described experimenter with the first therapeutic scheme, and second sub-group of described experimenter is treated with the second therapeutic scheme, the experimenter in the experimenter in wherein said first sub-group and described second sub-group has the average ratio of significantly different monomer A β than monomer and oligomer A β.In some method, experimenter's prevention of described first sub-group or the drug therapy for the treatment of A Zihai Mo's disease, the experimenter of described second sub-group need not described drug therapy, and compared with the experimenter in described second sub-group, monomer A β is than the ratio of monomer and oligomer A β, obviously lower in the experimenter of described first sub-group.

Present invention also offers the method for determining which experimenter in colony to be called up in clinical testing, described method comprises each experimenter in described colony: the amount measuring monomer A β in humoral sample; Measure the amount of monomer and oligomer A β in the second sample of body fluid; And the amount of the amount of monomer A β and monomer and oligomer A β is compared, wherein on basis of the comparison, the experimenter in described colony is called up or does not call up in described clinical testing.In some method, described comparison determines monomer A β and the ratio between monomer and oligomer A β, and the experimenter that wherein monomer A β falls within threshold value than the quotient of monomer and oligomer A β is called up in described clinical testing.

Present invention also offers a kind of diagnostic kit, it comprises: at least one terminal specificity holds antibody for the C-of A β 37, A β 38, A β 39, A β 40, A β 41 or A β 42; In conjunction with the N-end of A β and/or the antibody of central epi-position; And oligomers A β is transformed into the depolymerization agent of monomer A β.In some kit, described C-holds antibody terminal specificity for A β 40 or A β 42.Some kit comprises terminal specificity and holds antibody and terminal specificity to hold antibody for the C-of A β 42 for the C-of A β 40, provides the multiple ratio for disease assessment, to improve accuracy or the sensitivity of methods and results.

Present invention also offers the method that screening has the medicament of the activity of antagonism A Zihai Mo's disease, described method comprises: contacted with described medicament by the transgenic rodents model of A Zihai Mo's disease; The amount of monomer A β and the amount of monomer and oligomer A β in the body fluid of the transgenic rodents relatively contacted with described medicament; And use and describedly relatively determine whether described medicament has the activity that can be used for treating A Zihai Mo's disease.

Present invention also offers the method analyzing A β, described method comprises: (a) measures the amount coming from A β in the humoral sample of experimenter, and depolymerization agent process do not used by wherein said sample; B () measures the amount coming from A β in the humoral sample of described experimenter, the process of wherein said sample depolymerization agent; And (c) compare the amount measured in step (a) and (b).In some method, described in step (a) and (b) is measured the antibody using terminal specificity to hold for the C-of A β and carries out.In some method, the comparison in step (c) determines the ratio of the amount in step (a) and the amount in step (b), or the difference between amount in step (a) and step (b).Some method also comprises and described ratio or difference is used for the diagnosis of A Zihai Mo's disease or its neurological susceptibility in described experimenter, prognosis or monitoring, amount in step (a), than the higher difference between the amount in the lower quotient of the amount in step (b) or step (b) and the amount in step (a), indicates the deterioration of the higher neurological susceptibility of the described disease of generation of described experimenter, higher possibility that described disease exists or the state of an illness.

In some method, step (a) and (b) measure at least one in A β x-37, A β x-38, A β x-39, A β x-40, A β x-41 and A β x-42.In some method, step (a) and (b) at least measure A β x-40.In some method, step (a) and (b) at least measure A β x-42.In some method, step (a) and (b) at least measure A β x-40 and A β x-42.In some method, the amount of A β uses one or more terminal specificity to carry out for the C-end antibody of A β 37, A β 38, A β 39, A β 40, A β 41 or A β 42.In some method, one or more C-hold antibody to comprise terminal specificity for the antibody of A β 40 and the terminal specificity antibody for A β 42.In some method, A β is measured by the affine sandwich assay of immunity, and described determination method comprises one or more C-described and holds antibody and the another kind of antibody in conjunction with N-end and/or central epi-position.In some method, described depolymerization agent comprises guanidine hydrochloride, guanidinium isothiocyanate, urea, thiocarbamide, lithium perchlorate and/or potassium iodide, non-ionic detergent, polyglycol, polyvinylpyrrolidone, polyphenol and/or hexafluoroisopropanol.In some method, step (a) and (b) use identical determination method to measure the amount of A β.In some method, described humoral sample is CSF sample or blood sample.

Some method also comprises step (d) and is used in the diagnosis of A Zihai Mo's disease or its neurological susceptibility in described experimenter, prognosis or monitoring by described ratio or difference, amount in step (a), than the higher difference between the amount in the lower quotient of the amount in step (b) or step (b) and the amount in step (a), indicates the deterioration of the higher neurological susceptibility of the described disease of generation of described experimenter, higher possibility that described disease exists or the state of an illness.In some method, experimenter does not have cognitive impairment, and step (d) assesses the neurological susceptibility that A Zihai Mo's disease occurs described experimenter.In some method, described experimenter has mild cognitive impairment, and (d) assesses the neurological susceptibility that A Zihai Mo's disease occurs described experimenter.In some method, described experimenter has cognitive impairment, and step (d) comprise use step (c) comparison and other symptoms of described experimenter's illness and/or the combination of sign to provide the diagnosis of A Zihai Mo's disease.In some method, described experimenter had been diagnosed as before carrying out described method suffers from A Zihai Mo's disease, and step (d) provides the instruction of described disease stage.In some method, described experimenter is just accepting treatment or the prevention of A Zihai Mo's disease, and step (d) provides described experimenter to the instruction of the response for the treatment of.In some method, described method is carried out at set intervals, and the instruction providing the response to treatment more over time in step (c).In some method, described experimenter is just with the flat pearl monoclonal antibody treatment of the immunotherapy for A β such as hundred.

Before some method is also included in and carries out step (a) and (b), with the anti-idiotype for hundred flat pearl monoclonal antibodies, optionally with the described sample of JH11.22G2 process.Some method also comprises the amount measuring Tau or P-Tau in described sample, wherein Tau or P-Tau is relative to the increase of control value, provides the neurological susceptibility of generation A Zihai Mo's disease of described experimenter, the existence of A Zihai Mo's disease or the further instruction that sb.'s illness took a turn for the worse.Some method also comprises the nursing supplier described diagnosis, prognosis or monitoring being informed described experimenter or described experimenter.The experimenter of some such method in colony carries out, first sub-group of wherein said experimenter is treated with the first therapeutic scheme, second sub-group of described experimenter is treated with the second therapeutic scheme, and compared with the experimenter of described second sub-group, the ratio of amount ratio amount of the A β of measurement in step (b) of the A β measured in step (a) is obviously lower in the experimenter of described first sub-group.In some method, described first therapeutic scheme comprises the medicine of prevention for A Zihai Mo's disease or treatment, and described second therapeutic scheme does not comprise described medicine.In some method, the ratio of amount ratio amount of the A β of measurement in step (b) of the A β measured in step (a), lower than threshold value in the experimenter of described first sub-group, and higher than described threshold value in the experimenter of described second sub-group.

Definition

Term " antibody " comprises complete antibody and binding fragment thereof.Usually, fragment and their complete antibodies of being derived from compete the specific binding with antigen.Fragment comprises the heavy chain of separation, light chain, Fab, Fab', F (ab') ₂, scFv, Diabody, Dab and nano antibody.Fragment is produced by recombinant DNA technology or by the enzyme of complete antibody or Chemical Decomposition.

Specific binding refers to that the amplitude detected of antibody (or other reagent) and target (component of such as sample) is higher and can with the combination occurring at least one non-specific binding had nothing to do on target and distinguish.Specific binding can be the result that particular functional group or particular space agree with key between (such as key type) and formed, and the result of non-specific binding normally Van der Waals force.But specific binding does not imply that reagent combines a kind of and only a kind of target.Therefore, reagent also can demonstrate the specific binding from the varying strength of several different target usually really, and only demonstrates non-specific binding with other targets.Specific binding generally includes 10 ⁷, 10 ⁸or 10 ⁹m ^-1or higher binding constant.

Term " epi-position " refers to the site of immunoglobulin (Ig) or antibody (or its Fab) specific binding on antigen.Epi-position usually from continuous amino acid or by the secondary of protein and/or three grades folding and both the non-contiguous amino acids be collocated in together are formed.The epi-position formed from continuous amino acid usually retains after being exposed to denaturing solvent, and is usually lost after with denaturing solvent process by secondary and/or three grades of epi-positions be folded to form.Epi-position comprises at least 3,4,5,6,7,8,9,10,11,12,13,14 or 15 amino acid usually in the space conformation of uniqueness.Determine that the method for the space conformation of epi-position comprises such as x-radiocrystallography and two dimensional NMR.See such as " the epitope mapping scheme in molecular biology method " (Epitope MappingProtocols in Methods in Molecular Biology), Vol.66, G.E.Morris edit (1996).

When antibody is called as in conjunction with epi-position in specific residue such as A β 1-11, its meaning is the polypeptide that antibody specific binding contains described specific residue (namely in this example A β 1-11).Such antibody must not contact each residue in A β 1-11.Displacement or the disappearance of each single amino acids in A β 1-11 also need not fixing loud binding affinities.

The epi-position (namely described epi-position comprises N-end or the C-terminal amino acid of described peptide) at most N-or the C-end place of end-specific antibodies specific binding A β peptide, but intensity combines lower or in the A β of the not more microscler formula of specific binding or form the residue of epi-position in APP.Therefore, terminal specificity means the A β peptide relative to terminating at 37,38,39,41,42 or 43 residues for the antibody of A β 40, and preferential (namely with the binding constant of at least high 10 times) is in conjunction with the antibody of A β 40.Similarly, terminal specificity means the A β peptide relative to terminating at 37,38,39,40,41 or 43 residues for the antibody of A β 42, is preferentially combined in the antibody of the A β peptide terminated at 42 residue places.

Term " experimenter " comprises the mankind and other mammalian subject.This term can refer to any individuality within never disease sign or symptom to the scope with whole disease symptoms.Individuality within the scope of this can from asymptomatic develop into there is disease one or more signs again to one or more symptoms with typical case (full blown) disease.The sign of disease and symptom can sequentially or occur simultaneously.The individuality being in these stages any can have or can not have the risk of heredity or the described disease of other known generations.

A Zihai Mo's disease can be diagnosed according to DSM-IV-TR criterion.

Mild cognitive impairment can be diagnosed according to American Psychiatry disease association's 2001 guides (2001guidelines of the American Academy of Neurology).In simple terms, these guides need the individual report to himself memory problems, preferably obtain other people confirmation; It is measurable higher than normal memory defects that the memory evaluation test of use standard detects; And carry out the normal general thinking of normal daily routines and reasoning skill and ability.

If individuality not yet suffers from the A Zihai Mo's disease of usual definition (such as by DSM IV TR), but have and described experimenter is placed in higher one or more known risks and assumptions (such as >70 year that the risk of described disease during determining time such as 5 year occur more obvious than general population, heredity, biological chemistry, family history, premonitory symptom and/or oligomer A β parameter disclosed herein), then described individuality has the high risk of A Zihai Mo's disease.

Neurological susceptibility refers to the possibility or the risk that disease occur and/or is about to occur described disease.Higher possibility or risk mean higher neurological susceptibility.Measure and indicate higher neurological susceptibility equally with the time shorter between seizure of disease.

Term " symptom " refers to the subjective evidence of the disease felt by experimenter, the gait such as changed." sign " refers to the objective evidence of the disease observed by doctor.

Statistically significant sexual cue p value≤0.05.

Diagnosis, prognosis or monitoring determination method usually determine in the current and future in experimenter or change wherein accurate lower than 100%, if but indicate from the information that described determination method obtains do not provide described information with described determination method situation compared with obviously higher or lower or the possibility of described situation occurs in the future, then remain useful.

Describe in detail

I. lead to and state

The invention provides the method that auxiliary A Zihai Mo's disease (AD) comprises its diagnosis developing into outbreak, prognosis and/or monitoring.Although practice of the present invention does not rely on the understanding of mechanism, it is believed that oligomer A β accounts for the signal portion of the solubility A β existed in the body fluid of AD patient, and major part is not detected by current method of immunity.It is believed that oligomer A β is the virulence factor of cognitive symptom in AD or the intermediate of amyloid plaques formation, described amyloid plaques itself is the virulence factor of the presentation of cognitive symptom in AD.The oligomer A β in body fluid can not be detected in former report, can explain suffer from and do not suffer from A Zihai Mo's disease experimenter body fluid in A β value between remarkable overlap.Method of the present invention can assess the oligomer content of A β in body fluid, and this assessment is used for diagnose AD, prognosis is provided for AD patient and/or in AD patient monitoring of diseases progress.Such assessment, for carrying out diagnosing and monitoring being made by conventional criterion the disease commitment before the diagnosis of A Zihai Mo's disease, is useful especially.

II.Aβ

A β is the chief component of the distinctive amyloid plaques of A Zihai Mo's disease.A β has several naturally occurring total length form (being directly obtained from amyloid precusor protein (APP) not degraded further by the cutting of β and gamma secretase).The modal Native full-length form of A β is called as A β 39, A β 40, A β 41, A β 42 and A β 43.The exemplary sequence of these peptides and they and their relation of large transmembrane glycoprotein APP of being derived from, illustrate in the Fig. 1 in Hardy etc., TINS20:155-158 (1997).

A β 42 has one sequence: NH ₂– Asp Ala Glu Phe Arg His Asp Ser GlyTyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly SerAsn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala – COOH (SEQ ID NO:1).

Native form A β 41, A β 40, A β 39, A β 38 and A β 37 and the difference of A β 42 are Ala, Ile-Ala and Val-Ile-Ala that they lack C-respectively and hold, Val-Val-Ile-Ala, Gly-Val-Val-Ile-Ala amino acid residue; With the difference of A β 42, A β 43 is that it is held at its C-and comprises additional Thr amino acid residue.These forms any can comprise the naturally occurring Polymorphic variant of above-mentioned sequence, such as Arctic sudden change.The clipped form of A β is in vivo by the further degraded (namely by the degraded outside β and gamma secretase) of A β or produced by the external degradation after obtaining humoral sample.Some naturally occurring A β fragment is held by brachymemma at N-.Up to the present the N-identified holds the example of the A β peptide of brachymemma to comprise the A β peptide with 6-42,11-40,11-43,12-43 or 17-40 amino acids residue.Up to the present other the naturally occurring A β fragments identified are to hold two ends to be punctured into feature from N-end and C-.The example of such peptide comprises the A β peptide with 3-34,6-27,6-34,6-35 or 11-34 amino acids residue.Other fragments of A β can obtain, although any degraded so preferably minimizes from the degraded the sample be separated.

Some technology for measuring A β differs the A β and fragment thereof that distinguish in humoral sample the total length form existed surely.Such as, a kind of antibody using terminal specificity to hold for the C-of A β 40 and specificity are for the immunoassay of the another kind of antibody of the central epi-position of 20-25 position residue, and can detect A β 40 and A β x-40 fragment, wherein x is about 1-20.Therefore, when measuring A β by such determination method, described determination method is in fact measured A β 40 and is had any fragment of A β x-40 form, and wherein x is about 1-20.Other determination methods only measure the A β of total length form substantially.Such as, a kind of antibody using terminal specificity hold for the C-of A β 40 and terminal specificity for N-end (such as in conjunction with 1-5 position residue epi-position or within it combine) the immunoassay of another kind of antibody, detection A β 40 and do not detect subfragrnent (exceeding background or negative control level).Other determination methods are quantitative mass spectral art such as, can measure the A β of total length form and fragment is measured on each ground in each ground.Because some determination method does not make differentiation between total length A β and its some fragment, therefore the denotion of A β is comprised to the total length A β and fragment thereof that exist in detected humoral sample, unless the essence of context, i.e. determination method separately has requirement.In simple terms, symbol A β x-y can be used for any fragment indicating A β peptide and terminate at y position residue, and wherein y can be 37,38,39,40,41,42 or 43.Such as, A β x-42 is used to indicate any fragment that total length A β 42 or 42 residue places of amino acid sequence of providing above terminate.Similarly, A β x-40 indicates total length A β 40 or its any fragment terminated at 40 residue places.

A β peptide and fragment thereof exist to represent the monomer of different aggregation extent, oligomer, protofibril and fibril form.No matter monomer A β means whether there is the A β that depolymerization solvent and reagent all have the monomer molecule amount of expection.Depend on length, the expection molecular weight of the monomer A β of total length form is about 3900 to 4700Da (such as A β 42 and A β 40 have the molecular weight of 4514 and 4330Da respectively).Clipped form depends on that length has the molecular weight of proportional reduction.Except additive method, molecular weight can be measured on gel, post (such as passing through HPLC) or mass spectrometer.Monomer A β also can lack change to identify by the molecular weight measured after with depolymerization agent process.Monomer A β also can functionally be defined as by with oligomer A β contrast the antibody that contrast prepared product that prepared product compares monomer A β shows the Preference of high at least 10 times, the A β of the antibody recognition that such as terminal specificity is held for the C-of total length form such as A β 40 or the A β 42 of A β.The prepared product of the fresh lysate of A β in DMSO mainly exists with monomeric form, and provides useful contrast for the molecular weight of evaluation test prepared product.A β leaving standstill several days and therefrom being isolated the prepared product of oligomer A β by the gel electrophoresis that is separated with oligomer by monomer or column chromatography such as size exclusion chromatography or immunoaffinity chromatography in water, can be used as the contrast of oligomer A β.

Oligomer A β refers at least two A beta molecules of noncovalent aggregates each other.It is believed that oligomer Α β holds the hydrophobic residue at (the membrane spaning domain part of APP) place to keep together by the C-of peptide at least in part.Be similar to monomer A β, oligomer A β is solvable in physiological conditions.Most of oligomer A β has about 2-20 or 5-20 A beta molecule.Oligomer A β can be identified by the molecular weight of at least dimer molecule.Such as, the oligomer of total length A β has the molecular weight at least about 7500Da.The oligomer of A β fragment may have the molecular weight lower than 7500Da, but great majority have the molecular weight higher than 4600Da.Oligomer A β can also be identified by the reduction of molecular weight after with depolymerization agent process.By depolymerization agent, all or most of oligomer A β in body fluid can be transformed into the monomer with the molecular weight being no more than about 4600Da.Under the depolymerisation conditions determined, great majority in body fluid or all oligomer A β are transformed into monomeric form, now using depolymerization agent to continue process can not make molecular weight occur change further, and/or there is not the detectable form with the distinctive molecular weight of oligomer.By some epi-position of the antibody recognition for monomer A β, especially C-end epi-position, can not detect in oligomer A β.This part that may be due to epi-position to sheltering completely, described in shelter by the physical bond between the single A β peptide forming oligomer A β, form oligomer A β single A β peptide in destroy the structural rearrangement of epi-position or both combinations cause.Therefore, the amount of oligomer A β functionally can be defined as the amount of monomer that (1) measure after with depolymerization agent process and oligomer A β and (2) without the monomer A β measured during depolymerization agent process amount between difference.

At intramolecular rearrangement gradually with after being polymerized further, oligomer A β produces the fibril aggregation that is thread and filamentous structure subsequently had more than 20 A β peptides and prolongation.Different from oligomer A β solvable in physiological conditions, filamentous A β is usually insoluble in physiological conditions.Because it is insoluble, filamentous A β is present in sediment such as amyloid plaques.Propose, the patch formed by filamentous A β may cause the cognitive defect relevant to A Zihai Mo's disease.Alternatively or in addition, the virulence factor of oligomer A β as A Zihai Mo's disease has been proposed.No matter oligomer A β is the intermediate of virulence factor or virulence factor, in the method for the invention its analysis be for diagnosing, the useful indicant of prognosis or monitoring.

A β 40 and A β 42 is the modal A beta forms existed in the mankind.Abundance ratio A β 42 height of A β 40 in blood and CSF about 10 times, but A β 42 is the principal modes existed in the A β assembled.Such as, and Roher etc. (Proc.Nat ' l Acad.Sci.USA 90:10836 (1993)) find, A β 42 accounts for 90% of A β in neuritic plaques, and accounts for 75% of A β in essence blood vessel sediment.In addition, A β 42 has the larger tendency forming oligomer in the solution, and A β 42 oligomer obviously forms fibril quickly than A β 40 oligomer.Bitan etc., Proc.Nat ' l Acad.Sci USA 100:330 (2003).Due to their relative abundance, the measurement of A β 40 or A β x-40 and/or A β 42 or A β x-42 in body fluid such as blood, serum, blood plasma or CSF, the substitute of the measurement of total solvable A β can be used as, and detect other forms of A β (such as A β x-37, A β x-38, A β x-39, A β x-41) without each.But method of the present invention comprises any type of A β (such as A β x-37, A β x-38, A β x-39, A β x-40, A β x-41, A β x-42) measurement alone or in combination.In the measurement of CSF, preferably at least measure A β 42 or A β x-42.In the measurement of blood, preferably at least measure A β 40 or A β x-40.

III. measure monomer and oligomer A β

Method of the present invention can measure the amount of oligomer A β in body fluid.This measurement is carried out preferably by the amount of the merging amount and monomer A β of measuring oligomer A β and monomer A β.Alternatively or in addition, method of the present invention directly can measure the amount of oligomer A β.Except other unit, amount can be measured with the unit of weight or binding signal.By using the calibration curve of known quantity analyte, the arbitrary unit of signal intensity can be transformed into weight.

Various technology can be used to the amount of the merging amount and monomer A β of measuring monomer and oligomer A β.Preferred technology comprises the affine determination method of quantitative immunological using antibody test target antigen.The combination using antibody is preferred, such as, in the affine determination method of the immunity of sandwich-type.Determination method preferably includes at least one capture antibody and at least one reporter antibody, described in catch and the different epi-positions on the same target molecule of reporter antibody identification.Determination method that quantitative immunological is affine, comprise sandwich assay, can be solid phase (such as ELISA or based on pearl (such as based on pearl)) or liquid phase (such as electrochemiluminescence).Determination method that quantitative immunological is affine is described in such as " antibody assay guide " (Antibodies:A Laboratory Manual) by generality, Harlow and Lane, Cold Spring Harbor Press, in Cold Spring Harbor, NY (1988).For detecting the example of the ELISA sandwich assay from the A β the sample that human experimenter obtains, be described in WO 99/27944 and United States Patent (USP) 7,700, in 309.Or, mass spectrometry or electrophoresis (such as kapillary or gel electrophoresis) can be used, then detected and quantitative monomer and/or oligomer A β by quantitative Western blot method, any one technology described is optionally such as, with immune affinity capture technology (such as immunoassays) and/or purified technology of protein (such as precipitating and/or chromatography, HPLC) is combined carries out.The analysis based on mass spectrometry of A β is described in such as Iurascu etc., Anal.Bioanal.Chem.395:2509 (2009), Portelius etc., Acta Neuropathol.120:185 (2010) and Wang etc., in J.Biol.Chem.271:31894 (1996).

For the measurement affine based on immunity of the amount of monomer A β in sample, at least one antibody used in determination method should distinguish monomer and oligomer A β.The antibody be applicable to comprises the antibody in conjunction with the epi-position in the C-end regions (i.e. 29-43 amino acids residue) of A β, is preferably the end-specific antibodies of C-end.Due to the conformation change that can distinguish monomer and oligomer A β and relative hiding peptide-peptide interaction and sterically hindered, some epi-position that monomer A β exists is not present on oligomer A β or is covered (masked) thereon, and specificity for the antibody of the epi-position in the C-end regions of monomer A β generally not in conjunction with oligomer A β.Therefore, such antibody allows substantially only to detect the monomer A β comprised in the sample of monomer and oligomer A β.C-hold end-specific antibodies can specificity for such as A β 37, A β 38, A β 39, A β 40, A β 41 or A β 42.The antibody (such as monoclonal antibody 21F12) that preferred C-hold end-specific antibodies to comprise antibody (such as monoclonal antibody 2G3) that specificity holds for the C-of A β 40 and specificity are held for the C-of A β 42.Such C-holds epitope-specific antibodies to hold epitope-specific antibodies (such as terminal specificity is for one or more antibody of A β 37, A β 38, A β 39, A β 40, A β 41, A β 42 or A β 43) to combinationally use individually or with one or more other C-, with in conjunction with the A β in sample.

In sandwich assay, distinguish that one or more antibody of monomer and oligomeric forms can be catch or reporter antibody, but be preferably one or more capture antibodies.Other one or more antibody used in such sandwich assay are in conjunction with epi-positions different from the epi-position that one or more distinguishing property antibody combine on monomer A β.For simplicity, one or more distinguishing property antibody are called as capture antibody, and are called as reporter antibody (but contrary specificity is also possible) in conjunction with one or more antibody of different epi-position.Such as, when C-holds epitope-specific antibodies to be used to catch monomer A β, central epi-position (in the residue of 12-28 position) specific antibody (such as monoclonal antibody 266) or N-hold epi-position (namely in the residue of 1-11 position) specific antibody (such as monoclonal antibody 3D6 or 10D5) to be used as reporter antibody.Use in conjunction with the antibody of central epi-position allows the N-detecting A β to hold the form of brachymemma, and these forms some or all of may not use N-to hold reporter antibody to detect.

For the measurement affine based on immunity of the merging amount of monomer in sample and oligomer A β, can by sample reagent (such as solvent) process oligomer A β being depolymerized to monomer A β.Then by level that the Sample Dilution of depolymerization tolerates the concentration of depolymerization agent to be reduced to immune affinity agent (namely catching and/or reporter antibody).The depolymerization agent tolerance of antibody can be determined by rule of thumb.Can use can depolymerization oligomer A β and do not suppress any reagent of the identification based on antibody of the monomer A β of depolymerization after suitable dilution.The depolymerization agent be applicable to comprises chaotropic agent, non-ionic detergent, solubilizer or lipophilicity reinforcing agent or its any combination (combination of such as chaotropic agent and detergent).For plan object oligomer A β being transformed into monomer, depolymerization agent can individually or with any effective combination, use with any effective ratio.The chaotropic agent be applicable to comprises such as guanidine hydrochloride, guanidinium isothiocyanate, urea, thiocarbamide, lithium perchlorate and potassium iodide.The non-ionic detergent be applicable to comprises series detergent, series detergent and series detergent.Other solubilizer/lipophilicity reinforcing agent comprises hexafluoroisopropanol and the polymkeric substance of size within the scope of 10,000 to 50,000Da (such as the polymkeric substance of polyglycol, polyvinylpyrrolidone, polyphenol).

The highest depolymerization agent concentration depends on the tolerance of immune affinity agent (namely catching and/or reporter antibody) and the sensitivity of method.Usually, the dilution of the about 1:5 to about 1:40 (such as about 1:5 to about 1:20 or about 1:10) of depolymerized sample will guarantee the depolymerization agent tolerance of antibody, and have minimum or not impact to the sensitivity of method.Therefore, if the highest of urea (or guanidine hydrochloride) can be determined to be 0.5M by tolerable concentration in immunoassay, and diluted by the sample 1:10 of depolymerization before immunoassay, the maximum concentration of the depolymerization agent of so allowing in depolymerized sample is 5M.For the combination of detergent, solubilizer/lipophilicity reinforcing agent (such as polymkeric substance) and solvent/depolymerization agent, similar analysis can be carried out.Such as, for the polymkeric substance of about 10,000 to about 40,000Da, maximum concentration can in the scope of about 5% to about 10%.

Sample is used depolymerization agent process, make A β all or substantially all in sample be in free state (namely further processing the signal in the determination method that can not be increased in detecting subsequently).In the sample of depolymerization agent process, the merging amount of monomer and oligomer A β can be measured by immunoassay, preferably sandwich assay.Owing to not needing to distinguish monomer and oligomer A β (namely owing to substantially there is not oligomer A β in the sample of depolymerization agent process) in the sample of depolymerization agent process, combination for combination not any antibody of overlapping epitope of A β therefore can be used as catching and reporter antibody.But, in order to the more direct comparability between determination method, determination method that will be identical with the amount being used for measuring monomer A β in sample (namely not yet with the sample of depolymerization agent process), is also preferably used for the merging amount that (putting into practice best) measures monomer and oligomer A β in the sample of depolymerization agent process.Therefore, such as, hold sandwich assay that epitope specificity reporter antibody is feature to measure the amount of monomer A β if used with C-end capture antibody and central authorities or N-, then preferably use and comprise identical C-and hold the identical sandwich assay of epitope specificity capture antibody and central authorities or N-end epitope specificity reporter antibody to measure the merging amount of monomer and oligomer A β.If use different determination methods to carry out two kinds of measurements, then can by reference to the measurement of the control sample of the monomer A β or monomer and oligomer A β with concentration known by measured value suitably normalization, to compensate the varying strength that antibody combines.

The measurement of the merging amount of monomer and oligomer A β, also can by realizing simple for the measurement separated of monomer A β (such as described above) and oligomer A β phase Calais.For based on the affine measurement of immunity, this can identify oligomer A β but the antibody of nonrecognition monomer A β is measured the amount of oligomer A β from the humoral sample that experimenter obtains and realized by using.Specificity not in conjunction with monomer A β is described in such as WO04/031400 for the antibody of oligomer A β.

Depend on the form of determination method, distinguishing between the monomer of monomer A β and merging and oligomer A β may not be absolute.In other words, what the antibody of preferred combination monomer A β may not be absolute compared with oligomer A β distinguishes, or the oligomer A β of 100% may can not be transformed into monomer A β with the process of depolymerization agent.In addition, due to protein or other macromolecular combinations with the epi-position of covering for distinguishing monomer and oligomer A β (such as C-holds epi-position), some is in fact that the A β of monomer may be rated as oligomer.Although lack complete accuracy, but before and after with depolymerization agent process, use the Immunoassays measure humoral sample comprising the antibody (C of such as terminal specificity holds antibody) of preferred combination monomer A β compared with oligomer A β, the acceptable substitute of the monomer of monomer A β and merging and the measurement of oligomer A β can be taken as, and therefore carry out data analysis subsequently.

By different way, described method can by carrying out with the Difference test with A β in the humoral sample without depolymerization agent process, and do not need by the substance characterization detected be monomer, oligomer, with the monomer of protein bound or other materials.In such method, the amount of A β is detected in the humoral sample coming from experimenter, depolymerization agent process not yet used by wherein said sample, and in another humoral sample coming from described experimenter, detect the amount of A β, depolymerization agent process used by wherein said sample, and the amount of the A β detected compared.Detect the antibody that uses terminal specificity to hold for the C-of A β or the another kind of antibody preferably in conjunction with monomer A β compared with oligomer A β carries out.Described ratio or the difference comparing the amount determining use and do not use the A β measured during depolymehzation step process.Described ratio or difference can be similar with the ratio or difference to monomer and oligomer A β mode be used for the diagnosis of A Zihai Mo's disease, prognosis or monitoring.Therefore, all discussion of the ratio of oligomer and monomer A β, quotient or difference and their measurement and explanation and the application to difference therapeutic scheme, be applicable to after doing necessary amendment in detail when existing and there is not depolymerization agent, use to utilize compared with oligomer A β preferably in conjunction with the A β of the Immunoassays measure of the antibody of monomer A β amount between ratio.Such as, do not use the amount of depolymerization agent and use the less quotient of the amount of depolymerization agent or use comparatively big difference between depolymerization agent and the amount not using depolymerization agent, indicating the higher neurological susceptibility of the generation disease of described experimenter, the higher possibility of disease existence or the deterioration of the state of an illness.Similarly, in additive method, tested population of subjects can be layered as the first and second sub-groups according to above-mentioned quotient or difference, and make described sub-group experience difference therapeutic scheme.Such as, have less quotient or can use prevention compared with the sub-group of big difference or treat the drug therapy of A Zihai Mo's disease, the sub-group with larger quotient or less difference can described drug therapy (comprise and do not accept treatment).

Preferably, the measurement of the merging amount of the amount of monomer A β and monomer and oligomer A β, at same sample, the different sample aliquot of same sample such as, is carried out.But measurement can be carried out on different sample, as long as there is rational basis to believe that sample is substantially the same, such as, when multiple sample was collected from the substantially the same sequence of positions of same experimenter in the substantially the same time.The measurement of the merging amount of the amount of monomer A β and monomer and oligomer A β, can simultaneously or in a sequence, preferably use identical reagent and instrument to carry out.For the sample obtained from the experimenter accepting the passive immunotherapy (namely accepting the flat pearl monoclonal antibody of therapeutic antibodies such as hundred of specificity for A β) being used for A Zihai Mo's disease, such as, process with the reagent (such as neutralizing the anti-idiotype of hundred flat pearl monoclonal antibodies, JH11.22G2) of described therapeutic antibodies in optionally sample being used before carrying out immunoassay.Or determination method can use the antibody for central authorities and C-end regions to carry out with reporter antibody as catching.Hundred flat pearl monoclonal antibodies do not disturb such determination method because it with away from central authorities or C-hold the site of antibody to be combined.

IV. specificity is for the antibody of A β

For detect A β antibody can by approximate classify be hold in conjunction with the N-of A β, central authorities or C-hold epi-position.N-holds epi-position to be 1-11 position residue, and central epi-position is 12 to 28 residues, and C-holds epi-position to be that 29 residues are to C-end (such as 37,38,39,40,41,42 or 43 residues).Antibody in conjunction with the epi-position in the C-end regions of A β comprises such as antibody 2G3,21F12 and 369.2B.Antibody in conjunction with the epi-position in the middle section of A β comprises such as antibody 266,15C11,2B1,1C2,4G8 and 9G8.Antibody in conjunction with the epi-position in the N-end regions of A β comprises such as antibody 12B4,12A11,6C6,3A3,2H3,10D5 and 3D6.

2G3 is the mAb holding epitope specificity to be combined with the C-being arranged in mankind A β, is specially and combines (Johnson-Wood etc., PNAS February 18,1997vol.94,1550-1555) at the C-end place of A β 40.

21F12 is the mAb holding epitope specificity to be combined with the C-being arranged in mankind A β, is specially and combines (Johnson-Wood etc., PNAS February 18,1997vol.94no.41550-1555) at the C-end place of A β 42.

369.2B is the mAb holding epitope specificity to be attached to the C-being arranged in mankind A β, is specially and combines at the C-end place of A β 42.369.2B antibody and variant thereof are described in such as US5, and 786, in 180.

Terminal specificity holds other antibody a large amount of of epi-position to be described in scientific literature neutralization/or commercially available (see such as Horikoshi etc. for the C-on a kind of mankind A β of form, Biochem.Biophys.Res.Commun.319,733-7 (2004), it refer to hybridoma 82E1,1A10 and 1C3, wherein the first terminal specificity for A β 40, second and the third specificity for A β 42; Iwatsubo etc., Neuron 13,45-53 (1994); Barelli etc., Mol.Med.3,695-707 (1997); Levites etc., J.Clin.Invest.116,193-201 (2006); WWW alzforum.org/res/com/an; Novos, Biologicals, catalog number (Cat.No.) NB300-225 (terminal specificity is for A β 40) and Autogen Bioclear catalog number (Cat.No.) ABT109 (terminal specificity is for A β 42)).

266 are and the central epi-position being arranged in mankind A β, are specially the mAb of 16-24 position residue specific binding.266 antibody and variant thereof are described in such as US 20050249725 and WO01/62801.Produce the clone of 266 monoclonal antibodies, be deposited in ATCC on July 20th, 2004 according to the clause of Budapest pact (Budapest Treaty), preserving number is PTA-6123.

15C11 is and the central epi-position being arranged in mankind A β, is specially the mAb of the mAb of 19-22 position residue specific binding.15C11 antibody and variant thereof are described in such as United States Patent (USP) 7,625,560 and WO 2006/066049.Produce the clone of 15C11 monoclonal antibody, be deposited in ATCC on Dec 13rd, 2005 according to the clause of Budapest pact (Budapest Treaty), preserving number is PTA-7270.

2B1 is and the central epi-position being arranged in mankind A β, is specially the mAb of 19-23 position residue specific binding.2B1 antibody and variant thereof are described in such as US 20060257396 and WO2006/066171.Produce the clone of 2B1 antibody, be deposited in ATCC on November 1st, 2005 according to the clause of Budapest pact (Budapest Treaty), preserving number is PTA-7202.

1C2 is and the central epi-position being arranged in mankind A β, is specially the mAb of 16-23 position residue specific binding.1C2 antibody and variant thereof are described in such as US 20060257396 and WO2006/066171.Produce the clone of 1C2 antibody, be deposited in ATCC on November 1st, 2005 according to the clause of Budapest pact (Budapest Treaty), the registration number of appointment is PTA-7199.

9G8 is and the central epi-position being arranged in mankind A β, is specially the mAb of 16-21 position residue specific binding.9G8 antibody and variant thereof are described in such as US 7,625,560 and WO2006/066049.Produce the clone of 9G8 antibody, be deposited in ATCC on November 1st, 2005 according to the clause of Budapest pact (Budapest Treaty), the registration number of appointment is PTA-7201.

4G8 is and the central epi-position being arranged in mankind A β, is specially the mAb (Covance SIG-39220) of 17-24 position residue specific binding.

12B4 holds epi-position with the N-being arranged in mankind A β, is specially the mAb of 3-7 position residue specific binding.12B4 antibody and variant thereof are described in US20040082762 and WO03/077858.

12A11 holds epi-position with the N-being arranged in mankind A β, is specially the mAb of 3-7 position residue specific binding.12A11 antibody and variant thereof are described in such as US20050118651A1, US 20060198851, WO04/108895A2 and WO 2006/066089.Produce the clone of 12A11 monoclonal antibody, be deposited in ATCC on Dec 13rd, 2005 according to the clause of Budapest pact (Budapest Treaty), preserving number is PTA-7271.

6C6 holds epi-position with the N-being arranged in mankind A β, is specially the mAb of 3-7 position residue specific binding.6C6 antibody and variant thereof are described in such as US 20060257396 and WO2006/066171.Produce the clone of antibody 6C6, be deposited in ATCC on November 1st, 2005 according to the clause of Budapest pact (Budapest Treaty), the registration number of appointment is PTA-7200.

3A3 holds epi-position with the N-being arranged in mankind A β, is specially the mAb of 3-7 position residue specific binding.2H3 is specifically bound to the N-being arranged in mankind A β to hold epi-position, is specially the mAb of 2-7 position residue.3A3 and 2H3 antibody and variant thereof are described in such as US20060257396 and WO 2006/066171.The clone producing antibody 2H3 and 3A3 has ATCC registration number PTA-7267 and PTA-7269 respectively, on Dec 13rd, 2005 according to the clause of Budapest pact (Budapest Treaty) by preservation.

3D6 is specifically bound to the N-being arranged in mankind A β to hold epi-position, is specially the mAb of 1-5 position residue specific binding.Produce the clone (RB963D6.32.2.4) of 3D6 monoclonal antibody, American type culture collection (American Type CultureCollection (ATCC) is deposited according to the clause of Budapest pact (Budapest Treaty) on April 8th, 2003, Manassas, Va.20108, USA), preserving number is PTA-5130.10D5 is specifically bound to the N-being arranged in mankind A β to hold epi-position, is specially the mAb of 3-7 position residue.Produce the clone (RB4410D5.19.21) of 10D5 monoclonal antibody, be deposited in ATCC on April 8th, 2003 according to the clause of Budapest pact (Budapest Treaty), preserving number is PTA-5129.3D6 and 10D5 antibody and humanization thereof and chimeric versions thereof further describe in such as US 20030165496 and 20040087777 and WO02/088306, WO02/088307, WO02/46237 and WO04/080419.Other humanizations 3D6 antibody is described in US 20060198851 and WO 2006/066089.

Other antibody that can be used for the amount measuring monomer and/or oligomer A β in body fluid can be separated again.Antibody can stem from any applicable animal, comprises the immunity of rabbit, mouse, rat, cavy, hamster, goat, milk cow and chicken.Or antibody can by external system of selection such as phage display or by carrying out immunity to produce to transgenic mice, and described technology allows the antibody of other types, comprises human antibodies or nano antibody.

Antibody can be polyclone, monoclonal, chimeric or humanized.End-specific antibodies by order to wish specificity for A β end be termination small peptide (such as 4-8 amino acid) carry out immunity to manufacture.Such as, A β 38-42 peptide can be used as immunogene to produce the end-specific antibodies for A β 42, A β 37-41 can be used as immunogene to produce the end-specific antibodies for A β 41, A β 36-40 can be used as immunogene to produce the end-specific antibodies for A β 40, A β 35-39 can be used as immunogene to produce the end-specific antibodies for A β 39, A β 34-38 can be used as immunogene to produce the end-specific antibodies for A β 38, or A β 33-37 can be used as immunogene to produce the end-specific antibodies for A β 37.Described small peptide is connected to carrier to assist to cause immune response.Preferentially in conjunction with the ability of the A β of desired form A β, APP relative to more microscler formula of screening antibodies or their fragment, but the described fragment part comprised as longer protein do not have required terminal specificity for the immunogenic amino acid of free-end.Polyclone end-specific antibodies can by similar immunity, and removing lacks required specific antibody and manufactures on the affinity column of the more microscler formula of A β, APP or their fragment, described fragment comprise do not have required terminal specificity for the immunogenic amino acid of free-end.The antibody be applicable to and fragment thereof can recombinant production.In addition, other recombinant proteins (synbodies see such as described by WO/2009/140039) of the binding specificity of analog antibody can be used.

Specificity, for the antibody of A β, can use the immunity of the epi-position in the epi-position in the N-end regions (i.e. 1-11 amino acids residue) comprising required target epi-position such as A β, the epi-position in middle section (i.e. 12-28 amino acids residue) or C-end regions (i.e. 29-43 amino acids residue) originally to prepare.Carrier molecule can be coupled to immunogene, and for preparing antiserum or monoclonal antibody by routine techniques.The immunogene be applicable to has at least 5 continuous residues in A β usually, and may comprise more than 6 residues.Carrier molecule comprises seralbumin, keyhole limpet hemocyanin or other protein carriers be applicable to, as at Hudson and Hay, " practical immunology " (Practical Immunology), Blackwell ScientificPublications, Oxford, (1980), generally in the 1.3rd chapter to describe.

Depend on used measurement determination method, antibody can be modify or unmodified.Such as, capture antibody can be coupled to affinity reagent such as biotin, Avidin or small peptide (such as his-label).Then can by special high-affinity interact (combination of such as biotin and Avidin or streptavidin) affinity reagent is connected to solid substance.Solid substance can be such as pearl, or the surface in hole, and can use the interaction of the high-affinity of affinity reagent that capture antibody is attached to solid substance.Or, specificity can be adsorbed onto solid substance (such as plastics dish, pearl) for the second antibody of a part (such as constant region) for capture antibody, and for capture antibody is attached to solid substance.Similarly, can modify to comprise label to reporter antibody, or specificity can be used for the second antibody of a part (such as constant region) for reporter antibody to provide label.Label in reporter antibody or second antibody can be such as enzyme (such as connected by chemical linkers or with antibody frame in merge), fluorescence molecule, chemical illuminating reagent, chromophore, radioactive isotope or provide can any other chemicals of quantifiable signal or reagent.

V. sample

Method of the present invention measures the merging amount of monomer and oligomer A β, the amount of monomer A β and/or the direct amount measuring oligomer A β from the sample that experimenter obtains.Method can comprise and obtains sample and/or processing sample before carrying out the measurements from experimenter.Experimenter is generally the mankind, but also can be mammal such as rodent, is preferably mouse, such as, plays the transgenic mice of A Zihai Mo's disease model effect.Body fluid comprises such as cerebrospinal fluid (CSF), blood, urine and peritoneal fluid.Blood can mean whole blood and blood plasma or serum.

Sample preparation can comprise storage (such as in room temperature, at 4 DEG C or freezing) and/or the transport of sample.Other process can comprise such as by centrifugal blood to obtain blood plasma, or blood clotting is centrifugal to obtain serum.Further sample preparation, if present, depends on the determination method form of the amount for measuring monomer and/or oligomer A β, and can comprise biochemical steps such as protein precipitation and/or column chromatography.But, observe polystyrene collection tube in conjunction with A β, cause sample weight loss, and polypropylene tube has not shown similar A β binding affinity, and be preferred.

VI. the use of A β measured value

The original measurement value of amount of the monomer merged and oligomer A β (or oligomer A β) and monomer A β can be processed into information useful in the diagnosis of A Zihai Mo's disease, prognosis and monitoring.Described method provides the amount of monomer A β in body fluid and the merging amount of monomer and oligomer A β usually.This tittle can be compared, to provide several useful parameter of experimenter's situation.Preferably, the ratio between the amount of monomer A β and the merging amount of monomer and oligomer A β is determined.Described ratio can be expressed as monomer A β than the quotient of monomer and oligomer A β or in contrast (reciprocal number).Due to the inverse that reciprocal number is quotient, therefore the determination of ratio is considered to determine quotient and reciprocal number.Monomer A β is the tolerance that total A β in body fluid takes the mark of monomeric form than the quotient of monomer and oligomer A β.Deduct this mark with 1, provide the mark of oligomer A β in the total A β in body fluid.Quotient or mark also can be expressed as percent.Described amount also can be compared with the amount providing oligomer A β by the amount deducting monomer A β in the amount from monomer and oligomer A β.Or described amount can by determining that the ratio between monomer A β with oligomer A β compares.Compare by these parameter determined and be collectively referred to as the parameter relevant to oligomer A β.

The parameter relevant to oligomer A β is used to diagnose, prognosis or monitoring.Diagnosis, prognosis and monitoring may not be repelled mutually, because when being applied to the continuous system of morbid state and development, same parameters can use in a variety of different ways.Such as, parameter can indicate the present situation (diagnosis) and prediction future (prognosis) of experimenter.Parameter can provide Current Diagnostic and can be one of series of parameters used in monitoring.In general, in body fluid, the amount of oligomer A β increases, relevant to the possibility of the increase that the neurological susceptibility of the increase of the disease of described experimenter, disease exist or the deterioration of the state of an illness.Taking in ratio, especially body fluid monomer A β than the parameter of the form of the ratio between monomer and oligomer A β, is preferred for reducing due to distortion that the difference of A β total content in body fluid between subjects causes.The amount of oligomer A β increases, and reduces the quotient of monomer A β than monomer and oligomer A β.Therefore, monomer A β reduces than the quotient of monomer and oligomer A β, relevant to the possibility of the increase that the risk of the increase of the generation disease of described experimenter, disease exist or the deterioration of the state of an illness.Similarly, the increase of oligomer A β reduces the quotient of monomer A β than oligomer A β, and the deterioration of the possibility of increase that exists of the risk of the quotient and the increase of the generation disease of described experimenter that reduce, disease or the state of an illness is relevant.On the contrary, the increase of oligomer A β improves oligomer and monomer A β than the quotient of monomer A β or the oligomer A β quotient than monomer A β; The quotient of raising is relevant to the possibility of the increase that the risk of the increase of the generation disease of described experimenter, disease exist or the deterioration of the state of an illness.Monomer and oligomer A β relatively can otherwise process, and are similarly associated with the risk of the increase of the generation disease of described experimenter or reduction, increase that disease exists or the possibility of reduction or the improvement of the state of an illness or deterioration.

By the various parameters that the comparison of the measured value of monomer and oligomer A β is determined, can compare with baseline value, to assist the diagnosis of A Zihai Mo's disease, prognosis or monitoring.Baseline value can be the parameter value determined from the control group of experimenter.Control group can be negative control group or positive controls.The negative control group be applicable to is lower than 60 years old and does not have the individuality of any known genetic risk of any known sign of A Zihai Mo's disease or symptom or its.The positive controls be applicable to is the individuality being diagnosed with A Zihai Mo's disease.Or, the parameter value that baseline value obtains from same experimenter before can being.

In the negative control group of experimenter, monomer A β is contemplated to about 1.0 (such as about 0.90 to about between 1.10) than the baseline value of the quotient of monomer and oligomer A β.Quotient lower than the average quotient in negative control group indicates experimenter has increase neurological susceptibility to A Zihai Mo's disease, or has the possibility of the increase that A Zihai Mo's disease exists.There is between average quotient in quotient in experimenter and colony the difference of the statistically significant of at least 95% degree of confidence, particularly useful in formation diagnosis or prognosis.But less fiducial interval (such as between about 67 to 95% degree of confidence) is risky and start in the mensuration of other biological mark or the monitoring in time of quotient sign experimenter, is also valuable.Lower than the quotient (exceed experimental error, preferably use the confidence level estimation of at least 95%) of the former baseline value determined of experimenter, sb.'s illness took a turn for the worse to indicate experimenter.

The baseline (sometimes referred to as threshold value) of parameter also can define according to the former mensuration of test experimenter.Such as, in the population of subjects not having Symptomatic A Zihai Mo's disease, than the quotient of monomer and oligomer A β, and described colony can be followed the tracks of subsequently to determine that A Zihai Mo's disease occurs which experimenter by measurement parameter such as monomer.Then baseline or threshold value can be set to makes in known error span, the experimenter fallen within described threshold value has a kind of result (such as A Zihai Mo's disease occurring), and has another kind of result (such as keeping not having A Zihai Mo's disease) higher than the experimenter of described threshold value.There is the experimenter of the parameter value being just in time in threshold value place, be usually all assigned to a kind of result or another kind of result according to the setting means of threshold value, or can be assessed as uncertain.The probability of error and thing followed false positive and false-negative possibility, the value that can be set by threshold value is controlled.As another example, can be suffered from or the parameter value do not suffered from the colony of A Zihai Mo's disease sets threshold value, to determine the presence or absence of A Zihai Mo's disease by more known.Similarly, the exact value of threshold value can be configured to false positive and false-negative quantity to remain in permissible range.Permissible range is for may be different different health care practitioners, but preferably threshold value is selected such that false positive and/or false-negative quantity are less than 20%, are less than 15%, are less than 10% or be preferably less than 5%.Different threshold values can be used for different diagnosis, prognosis and monitoring.Preferably, the value of baseline and threshold value use with such as, for determining that the determination method form (such as identical determination method type and identical reagent, the identical specificity used in the affine sandwich assay of immunity is caught and reporter antibody) that ratio is identical is determined testing in experimenter.Similarly, baseline and the value of threshold value preferably use with for determining that the sample preparation technology that ratio is identical is determined testing in experimenter.

Come from the parameter of the comparison of the measuring amount of monomer and oligomer A β, usually with other signs of experimenter and the assessment of symptom, especially experimenter's cognitive ability and/or the level of other biological mark combined, assistance provides prognosis, diagnosis or monitoring information.ADAS-CO 11, ADAS-CO 12, DAASD, CDR-SB, NTB, NPI, MMSE are the known scales for assessment of cognitive function.Other biological mark comprises ^[18F]fDG, MRI mark (BBSI and VBSI), CSF biomarker A β 42, Tau and/or P-Tau, and the PET imaging of A β in brain.The sign of A Zihai Mo's disease and symptom in experimenter, if present, can determine the target analyzed.Such as, in asymptomatic experimenter, target is normally determined the neurological susceptibility of A Zihai Mo's disease and/or is monitored towards advancing of disease, if there is any sign, then carries out next step.In the experimenter with cognitive impairment, target can be determine the neurological susceptibility of generation A Zihai Mo's disease with monitoring towards advancing of disease, but target also can be the existence determining or get rid of A Zihai Mo's disease.Be diagnosed as in the experimenter suffering from A Zihai Mo's disease by other criterions (such as DSM-IV-TR), target can be the future development determined disease stage, confirm diagnosis or monitoring of diseases.In experimenter to be treated, target can be measure the response to treatment.

Therefore, such as, in the experimenter not demonstrating cognitive decline symptom, lower than the amount of the monomer A β of the baseline value of negative control group (as defined above) than the quotient of the merging amount of monomer and oligomer A β, indicate described experimenter has the increase that A Zihai Mo's disease occurs neurological susceptibility relative to control population.For same experimenter, lower than the amount of the monomer A β of the former quotient determined of described experimenter than the quotient of the merging amount of monomer and oligomer A β, indicate towards the development of A Zihai Mo's disease.

For the experimenter demonstrating mild cognitive impairment (MCI) symptom, lower than the amount of the monomer A β of the specific baseline value of negative control experimenter than the quotient of the merging amount of monomer and oligomer A β, indicate the neurological susceptibility that described experimenter has the raising that A Zihai Mo's disease occurs.Mild cognitive impairment itself is the illness confirmed, and may be the prodromic phase of A Zihai Mo's disease, but also may occur due to other reasons.Therefore, the combination of lower quotient and MCI symptom, indicates and has MCI with normal quotient or have identical quotient and do not have the experimenter of MCI to compare the neurological susceptibility of the raising of A Zihai Mo's disease.For the experimenter with MCI, indicated towards the development of A Zihai Mo's disease lower than the former quotient to the quotient that described experimenter determines.

For the experimenter demonstrating the comprehensive cognitive decline symptom no matter whether being categorized as MCI, described decline may occur relevant to A Zihai Mo's disease or its, or may be irrelevant dementia.In such individuality, lower than the amount of the monomer A β of the baseline value of negative control experimenter than the quotient of the merging amount of monomer and oligomer A β, optionally with other signs and the symptom combination of disease, can be used for diagnosis or get rid of A Zihai Mo's disease.

For being diagnosed as the experimenter suffering from A Zihai Mo's disease, lower than the amount of the monomer A β of threshold value than the quotient of the merging amount of monomer and oligomer A β, can be used for carrying out classification to illness.Such as, threshold value can be defined as the moment (such as slightly, moderate, late period) corresponding to A Zihai Mo's disease.Lower than the quotient of the quotient determined for described experimenter in the past, the state of an illness indicating experimenter worsens.Therefore, quotient can be used to monitor the situation of experimenter.Such as, if experimenter is just accepting A Zihai Mo's disease therapy (such as immunotherapy, hundred flat pearl monoclonal antibody immunotherapies), then can use quotient to monitor the response to therapy.Quotient depends on healing potion over time.For immunotherapy, along with the A β sediment in brain is dissolved and is discharged into body fluid, healing potion may cause quotient in body fluid reducing at the beginning.But finally, along with oligomer A β removes from body fluid, described quotient may increase.Such as suppress in the Small molecular of A beta peptide aggregation at other medicaments, described quotient may respond to successful treatment and increase, and does not have temporary transient reduction.

Be for purposes of illustration to the amount of monomer A β than the discussion of the quotient of the merging amount of monomer and oligomer A β, but above-mentioned any parameter can additionally or alternatively use in a similar manner.Certainly, in method, there are some surface differences.Such as, when using the quotient of monomer A β and oligomer A β than monomer A β, the value higher than (instead of lower than) specific baseline or threshold value indicates experimenter to be had or is easy to the illness relevant to A β occurs.The baseline value expection produced from negative control population of subjects can be about 1.0 (such as about 0.95 to about between 1.10).For the amount of oligomer A β, the threshold value of instruction A Zihai Mo's disease or the neurological susceptibility to this illness can be about 0.3ng/mL.

The amount of Tau or phosphorylation Tau (i.e. P-Tau) in the sample coming from experimenter is that the parametric joint can calculated to the gauge from monomer and oligomer A β uses to assist the diagnosis of the illness relevant with A β or prognosis or assists to monitor the preferred biomarker of the illness of being correlated with A β.Tau is microtubule bindin (Goedert etc., the Neuron 3:519-526 (1989) existed in the NFT in A Zihai Mo's disease patient brain; Goedert, TINS 16:460-465 (1993)).The increase of Tau, especially P-Tau level in CSF, is associated with neure damage and A Zihai Mo's disease.Such as, the Tau of about 300pg every milliliter in CSF, can as the threshold indicator suffering from A Zihai Mo's disease, wherein exceed or equal the amount of Tau of 300pg/mL in CSF, indicate the higher possibility that experimenter has or is easy to occur the illness relevant to A β, and lower than the amount of the Tau of 300pg/mL in CSF, indicate experimenter do not have or be not easy to the generation illness relevant to A β higher possibility (see United States Patent (USP) 7,700,309).

Tau can be detected by such as immunoassay.Useful detection technique comprises such as immune affine sandwich assay, and it comprises the reporter antibody (see United States Patent (USP) 7,700,309 and PCT/US11/033649) of equal specificity for the capture antibody of Tau and mark.Antibody for Tau is commercially available (such as from Sigma, St.Louis, MO), or known (United States Patent (USP) 7,700,309), or can be prepared by conventional method.

Method of the present invention may need obtain from experimenter or receive humoral sample, the amount measuring one or more analytes (such as monomer A β and monomer add oligomer A β) is measured to sample, data analysis is carried out to provide diagnosis, prognosis or monitoring information to the value measured, and by described information interchange to experimenter, caretaker or hygiene care supplier.In some method, undertaken by the one or more individualities in same entity (such as medical institutions, hospital or hygiene care tissue) in steps.Or method can be undertaken by the individuality coming from the different entities working under contract or otherwise cooperate.Such as, the individuality in an entity can be ordered determination method and obtain Samples subjects, and by information interchange to experimenter or caretaker.Individuality in another tissue can carry out determination method and some or all of data analysis.

VII. computer-implemented

One or more steps (except wet chemistry step) of method can be carried out in the computing machine of suitably programming.The calculating of one or more parameters relevant to oligomer A β can be carried out in such computing machine.Come from any A beta form (monomer, monomer add oligomer, use or without denaturing solvent process) the raw data of measurement, calibration curve original signal be associated with numerical value such as stored in a computer can be used, be processed into numerical value (such as amount or concentration) in a computer.Computing machine also can be programmed, with provide the value of the actual measured amount of the A β taking any detected form, the parameter relevant to oligomer A β, patient situation (such as diagnose, prognosis, monitoring, disease progression, generation A Zihai Mo's disease risk) and/or treat the output of option.

The present invention can implement in hardware and/or software.Such as, different aspect of the present invention can be implemented in user side logical OR server end logic.The present invention or its ingredient can be embodied in the fixed mediums program assembly containing logical order and/or data, and described instruction and/or data when in the computer installation being loaded into suitably configuration, make described device perform according to the present invention.The observer that the fixed mediums containing logical order can be delivered to fixed mediums with physical loading in the computing machine of observer, or the fixed mediums containing logical order can reside in far-end server, observer enters described server to download assembly by medium of communication.

Hardware can be personal computer or anyly apply mutual massaging device with remote data, such as digital to television, cell phone or personal digital assistant.Reside in Information Availability in primary memory or supplementary storage in the such system of programming, and can show as the light of dish type or magnetic medium, tape, solid-state dynamically or static memory etc.Such as, the present invention all or part ofly can be presented as the software be recorded in such fixed mediums.The various programs be stored on primary memory can comprise receive to multiple A beta form (monomer, monomer add oligomer, use or without denaturing solvent process) the program of the relevant signal of measurement, such signal is processed into the program of numerical value (such as amount or concentration), calculate the program of the value of the parameter relevant to oligomer A β from these actual measured amount, explain the program etc. of the parameter relevant to A β according to experimenter's situation, prognosis or treatment plan.Such program can partially by the one or more calculated values by parameter relevant to oligomer A β in experimenter, and the database with the storage of the such value relevant to the situation of experimenter, prognosis or treatment plan compares work.Computer memory also can stored routine, with provide the value of the actual measured amount of the A β taking any detected form, the parameter relevant to oligomer A β, patient situation (risk of A Zihai Mo's disease such as occurs) and/or treat the output of option.Output can be provided on such as display or be stored into other memory storages (such as ZIP dish, CD-R, DVD, floppy disk, flash card) and/or print to hard copy such as on paper.The result of process can store in whole or in part or show, and this is determined by user.

VIII. clinical testing

The parameter relevant to oligomer A β determined above, can be used for determining whether experimenter to call up in clinical testing.Described clinical testing can be used for test to the prevention of A Zihai Mo's disease or may treat useful medicine.Described medicine can be such as antibody (such as specificity is for the antibody of A β) or be designed to induce for the immunogene of the antibody of A β.

The parameter relevant to oligomer A β and the threshold value be applicable to are compared.The threshold value be applicable to depends on the used parameter relevant to oligomer A β and test objective.Such as, threshold value can be selected to only qualification and has the experimenter of the high likelihood suffering from A Zihai Mo's disease, or it can be selected to qualification has high neurological susceptibility experimenter to A Zihai Mo's disease.There is in colony the experimenter of the parameter relevant to oligomer A β higher or lower than threshold value, possess the qualification participating in clinical testing.Such as, there is amount lower than the monomer A β of applicable threshold value than the experimenter of the quotient of the merging amount of monomer and oligomer A β in colony, possess the qualification participating in clinical testing, and there is in colony the experimenter of the quotient higher than threshold value, do not possess the qualification participating in clinical testing.Or, have higher than the oligomer of applicable threshold value and the monomer A β experimenter than the reciprocal number of monomer A β or the amount of oligomer A β in colony, possess the qualification participating in clinical testing, and the experimenter of the such quotient had in colony lower than threshold value or amount, do not possess the qualification participating in clinical testing.Use the parameter relevant to oligomer A β to create as the criterion of calling up experimenter in clinical testing evenly colony, wherein there is not or exist the individuality of less shortage A Zihai Mo's disease or the high neurological susceptibility to disease, and unlikely demonstrate the false positive of the treatment that will test is responded.

IX. the therapeutic scheme changed

The parameter relevant to oligomer A β determined above also can be used for determining which experimenter accepts or do not accept therapeutic scheme.Such parameter and the threshold value be applicable to are compared.According to described comparison, can to snibject's medicine to implement prevention or the treatment of A Zihai Mo's disease.Or for the experimenter accepted for the prevention of the illness relevant to A β or the medicine for the treatment of, the described dosage that experimenter relatively can be indicated just to accept increase, reduce or cancel and replace different pharmaceutical.

Such as, do not accept any treatment of A Zihai Mo's disease or the experimenter of prevention, can be classified as to have monomer A β higher or lower than threshold value than the quotient of monomer and oligomer A β, experimenter wherein lower than threshold value accepts treatment or prevention subsequently, and the experimenter being equal to or higher than threshold value does not accept treatment or prevention.Accept the experimenter for the prevention of A Zihai Mo's disease or the medicine for the treatment of, the monomer that can merge according to monomer A β ratio and the quotient of oligomer A β are higher than still classifying lower than threshold value, experimenter wherein higher than threshold value continues to accept medicine, experimenter lower than threshold value carries out the adjustment of drug dose, or the novel drugs of the prevention changed into for A Zihai Mo's disease or treatment.Or, for the experimenter accepting medicine, the baseline value of quotient with the quotient corresponding to the identical parameters determined for described experimenter in the past can be compared.If quotient is lower than baseline value, increasing dose or experimenter the novel drugs of prevention for A Zihai Mo's disease or treatment can be changed into.If quotient is higher than baseline value, the drug dose of experimenter can reduce or remain unchanged.

Other parameters relevant to oligomer A β, such as monomer and the oligomer A β quotient (reciprocal number) than monomer A β or the amount of oligomer A β, also can be used for changing in the method for the therapeutic scheme of the experimenter having the illness relevant to A β or be easy to occur the illness of being correlated with A β.Described method and above-described method similar, difference is when the amount of quotient or oligomer A β and threshold value being compared, can cause for experimenter's (such as not accepting the experimenter of any treatment) outputs medicine to implement prevention or the treatment of A Zihai Mo's disease higher than the quotient of threshold value or baseline value or amount, improve the drug dose of experimenter, or make experimenter change as the prevention of A Zihai Mo's disease or the novel drugs for the treatment of.Can cause reducing the drug dose of experimenter lower than threshold value or the reciprocal number of baseline value or the amount of oligomer A β or preservation dose constant.

Any one in the parameter relevant to oligomer A β that monomer A β and monomer are determined with the comparison from these values with the amount of oligomer A β, also can be used for determining any experimenter be applied in colony by two or more therapeutic schemes.Use the parameter relevant to oligomer A β that colony is layered as the first and second sub-groups, wherein relevant to A β between described colony parameter has the difference of statistically significant.Such as, in the first sub-group, monomer A β is different from the mean value of ratio described in the second colony with the difference of statistically significant than the mean value of the ratio of monomer and oligomer A β.Experimenter in first sub-group is treated with the first therapeutic scheme, and the experimenter in the second sub-group is treated with the second therapeutic scheme being different from the first therapeutic scheme.Therapeutic scheme can be short side case (namely experimenter does not accept treatment).Therefore, such as, experimenter in first sub-group can accept the medicine of prevention for A Zihai Mo's disease or treatment, and the experimenter in the second sub-group can not accept medicine (or at least not accepting the medicine identical with the experimenter in the first sub-group).When such as compared with the second sub-group, when the monomer of A β is lower than the ratio of monomer and oligomeric forms in the experimenter of the first sub-group, provide such difference scheme.Or the experimenter in the first sub-group can accept the first medicine of prevention for A Zihai Mo's disease or treatment, and the experimenter in the second sub-group can accept the such medicine of the second.Or the experimenter in the first and second sub-groups can accept the treatment for the various dose of the prevention of A Zihai Mo's disease or the same medicine for the treatment of, frequency or route.By some colony's layering, all experimenters in a sub-group are had be equal to or higher than the value of the parameter relevant to oligomer A β of threshold value, and all experimenters in another sub-group has the value of the parameter relevant to oligomer A β being equal to or less than threshold value.Other places here and in the application, there is the experimenter of the parameter value accurately equaling threshold value, usually be all assigned to a sub-group or another sub-group according to the setting means of threshold value, or be rated as uncertain and be not included in arbitrary colony.The number for the treatment of experimenter in colony and sub-group thereof should be enough to make between sub-group, and the difference of one or more parameter relevant to oligomer A β reaches the degree of statistically significant.Such as, described method can be applied to and comprise at least 20,50,100,1000 or 10, the colony of 000 experimenter.

Present invention also offers the method for experimenter being carried out to difference treatment in the sub-group of layering described above.Experimenter in different sub-group, the same medicine of prevention or the treatment accepting or do not accept for A Zihai Mo's disease can be passed through, by accepting to be used for the prevention of A Zihai Mo's disease or the different pharmaceutical for the treatment of, or different dosing dosage, frequency or the approach by accepting to be used for the prevention of A Zihai Mo's disease or the same medicine for the treatment of, carry out difference treatment.

X. kit

Present invention also offers for perform assist A Zihai Mo's disease diagnosis, prognosis and monitoring the kit of determination method.Kit can comprise and can be used for measuring sample, such as two or more A β specific antibodies of the amount of monomer A β from the body fluid that experimenter obtains.Antibody can such as can be used for carrying out immune affine sandwich assay.Preferably, kit comprises specificity at least one capture antibody of A β and the specificity at least one reporter antibody for A β, and described reporter antibody can be incorporated into monomer A β with described at least one capture antibody simultaneously.Any one at least one capture antibody or at least one reporter antibody is selected to specific binding monomer A β and can not in conjunction with oligomer A β.Such as, described at least one capture antibody can comprise the antibody holding epi-position in conjunction with C-, and described at least one reporter antibody can comprise the antibody in conjunction with N-end and/or central epi-position.Or described at least one capture antibody can comprise the antibody in conjunction with N-end and/or central epi-position, and described at least one reporter antibody comprises the antibody holding epi-position in conjunction with C-.The antibody be applicable to comprises any antibody described herein, comprises any fragment of such antibody.Preferred C-holds epitope-specific antibodies to comprise terminal specificity for the antibody (such as mAb 2G3) of A β 40 and the terminal specificity antibody (such as mAb21F12) for A β 42, but to replace or except the antibody of terminal specificity for A β 40, A β 42, terminal specificity can be comprised for all in A β 37, A β 38, A β 39 or A β 41 or one or more antibody of any one.Preferred central epitope-specific antibodies comprises the antibody (such as mAb 266) of specificity for the epi-position in the 12-28 amino acids residue of A β.N-holds epitope-specific antibodies to comprise specificity for the epi-position in the 1-11 amino acids residue of A β, is preferably the antibody of the epi-position in the 3-7 amino acids residue (such as mAb 10D5) of A β or the 1-5 amino acids residue (such as mAb 3D6) of A β.

Capture antibody in kit is optionally coupled to affinity reagent such as biotin, Avidin or peptide tag (such as his-label).Or kit can comprise the second antibody of specific binding capture antibody.Reporter antibody in kit is optionally coupled to label such as enzyme, fluorescence molecule, chemical illuminating reagent, chromophore, radioactive isotope or provides can any other chemicals of quantifiable signal or reagent.Or kit can comprise specific binding reporter antibody and comprise the second antibody of applicable label.

Kit of the present invention may further include the depolymerization agent (such as solvent) being suitable for depolymerization oligomer A β.Depolymerization agent can be such as any depolymerization agent described herein.Kit of the present invention also can comprise for blocking the reagent coming from the therapeutic antibodies existed in the sample of experimenter.Such as, blocking reagent can be anti-idiotype, and such as specificity is for the anti-idiotype (such as mAb JH11.22G2) of hundred flat pearl monoclonal antibodies.The inclusions that kit of the present invention can also comprise use kit is measured with the merging of the measurement or monomer and oligomer A β of carrying out measurement described herein such as monomer A β, or determines the instructions of the parameter relevant to oligomer A β such as above-described ratio.

XI. transgenic animals determination method

Many animal models (see such as WO93/14200, United States Patent (USP) 5,604,102,5,387,742 and 6,717,031) of A Zihai Mo's disease are reported.Useful especially animal model for A Zihai Mo's disease comprises mammalian animal model, is more specifically rodent model, especially muroid and Hamster model.Such animal model can comprise encodes and expresses the transgenosis of mankind APP or its fragment.Mankind APP transgenosis can be included in animal model and promote or accelerate the sudden change that A Zihai Mo's disease occurs.Described sudden change can be such as relevant to the mode of inheritance of A Zihai Mo's disease.Such as, suddenly change relevant to London or Indiana familial A Zihai Mo's disease, the sudden change at the 717 amino acids places of APP or Swedish sudden change (i.e. asparagine ⁵⁹⁵-leucine ⁵⁹⁶).Such sudden change is described in such as U.S. Patent number 7,700,309 and 6,717, in 031.These models can be used for screening compounds affects the process of A Zihai Mo's disease to improve and to aggravate the ability of illness.Because A Zihai Mo's disease increases to feature with the amount of the reduction of the amount of monomer A β in body fluid and oligomer A β, therefore the parameter relevant to oligomer A β is changed to effective treatment of A Zihai Mo's disease.Such as, the amount that the medicament accelerating A Zihai Mo's disease process tends to reduce monomer A β in sample is than the quotient of the merging amount of monomer and oligomer A β.On the contrary, slow down or stop amount that medicament that A Zihai Mo's disease is in progress may tend to improve monomer A β in the sample quotient than the merging amount of monomer and oligomer A β, although temporary transient reduction may be there is before any raising.Such test compound comprises antibody or its fragment, protein, little organic compound etc.

Method comprises transgenic animal model test compound being delivered medicine to A Zihai Mo's disease, measure the merging amount coming from monomer A β and oligomer A β and monomer A β in the humoral sample of animal, measure monomer A β and measure oligomer A β, or measuring oligomer A β simply; And one or more parameters relevant to oligomer A β of animal are determined according to described measurement.Depend on the determined parameter relevant to oligomer A β, the increase of the parametric statistics data compared with relevant baseline value or reduction, indicate test compounds and improve or increase the weight of A Zihai Mo's disease.Baseline value can be determined from the control-animal group of not yet reception test compound (such as similar or consistent heredity animal groups).

Such as, method can comprise the amount measured and come from monomer A β in the body fluid of animal, measure the merging amount of monomer and oligomer A β in body fluid, determine the quotient of the actual measured amount of the monomer A β of body fluid than the actual measurement merging amount of monomer and oligomer A β, and described quotient and the baseline value be applicable to are compared.If quotient is higher than baseline value, test compound is accredited as to the prevention of A Zihai Mo's disease or may treat useful medicine.Or if quotient is lower than baseline value, test compound is accredited as the medicine increasing the weight of or accelerate A Zihai Mo's disease progress.

Or, method can comprise the amount measured and come from monomer A β in the body fluid of animal, measure the merging amount of monomer and oligomer A β in body fluid, determine the monomer of body fluid and the actual measurement merging amount of the oligomer A β reciprocal number than the actual measured amount of monomer A β, and described reciprocal number and the baseline value be applicable to are compared.If quotient is lower than baseline value, test compound is accredited as to the prevention of A Zihai Mo's disease or may treat useful medicine.Or if quotient is higher than baseline value, test compound is accredited as the medicine increasing the weight of or accelerate A Zihai Mo's disease progress.

Or method can comprise the amount measured and come from monomer A β in the body fluid of animal, measure the merging amount of monomer and oligomer A β in body fluid, determine the amount of oligomer A β in body fluid, and the amount of oligomer A β and the baseline value be applicable to are compared.If described amount is lower than baseline value, test compound is accredited as to the prevention of A Zihai Mo's disease or may treat useful medicine.Or if described amount is higher than baseline value, test compound is accredited as the medicine increasing the weight of or accelerate A Zihai Mo's disease progress.Such method also can be undertaken by the amount directly measuring oligomer A β.

XII. version

Shang Mian Dui A Zihai Mo's disease and the same principle described by A β and strategy, can be used for other short amyloidosis (amyloidogenic) disease and component peptides thereof after suitable amendment.In other words, in body fluid, monomer is urged amyloidosis peptide and is added that monomer urgees the ratio (or as discussed above other correlation parameters) of amyloidosis peptide than oligomer, be used to provide the diagnosis of experimenter, prognosis or monitoring, wherein than oligomer, monomer adds that the relatively low quotient that monomer urgees amyloidosis peptide indicates the existence of the disease of described experimenter or the deterioration of neurological susceptibility or the state of an illness.Some examples of short amyloidosis diseases and component peptide thereof are: diabetes B, IAPP (Amylin); Parkinson's disease and other lewy body diseases, alpha-synapse nucleoprotein; Propagability spongiform encephalopathy (such as BSE), PrPSc; Heng Tingdunshi chorea, Huntington protein; Medullary carcinoma of thyroid gland, calcitonin (ACal); Arrhythmia cordis and isolated atrial amyloid, ANF (AANF); Atherosclerotic, apolipoprotein AI (AApoA1); Reactive amyloidosis, familial Mediterranean fever, familial amyloid nephropathy with nettle rash and deafness, and rheumatoid arthritis, serum amyloid sample peptide A (AA); Amyloid pathology inside sustainer, medin (AMed); Prolactinoma, prolactin (APro); Familial amyloid polyneuropathy, transthyretin (ATTR); The non-neuropathic SA of heredity, lysozyme (ALys); The amyloidosis relevant to dialysis, beta-2 microglobulin (A β 2M); Amyloidosis Finnish type, gelsolin (AGel); Lattice corneal dystrophy, Keratoepithelin (Aker); Cerebral amyloid angiopathy (Iceland's type), cystatin (ACys); Systematicness AL amyloidosis or Huppert's disease, light chain immunoglobulin AL; Sporadic occlusion body myositis, S-IBM; With the dyscrasic Heavy chain amyloidosis of several immunocyte.Other examples of short amyloidosis diseases and their peptide are provided in United States Patent (USP) 6,936, in the table 1 of 246.

Although for the object of clear understanding to invention has been detailed description, some amendment can be put into practice in the scope of claims.The all publications quoted herein and patent documentation with its full content be all objects by reference to being incorporated to herein, its degree is equal to and so describes individually each described document.Unless can as apparent from context, otherwise any step, feature, aspect, key element or embodiment can combination with one another use.

Claims

1. a method for the diagnosis of auxiliary A Zihai Mo's disease or its neurological susceptibility, prognosis or monitoring, described method comprises

A. the amount coming from monomer A β in the humoral sample of experimenter is measured;

B. the amount coming from monomer and oligomer A β in second humoral sample of described experimenter is measured;

C. the amount of the amount of described monomer A β and monomer and oligomer A β is compared; And

D. compare described for the diagnosis of A Zihai Mo's disease or its neurological susceptibility in described experimenter, prognosis or monitoring.

2. the method for claim 1, wherein step (c) determines monomer A β and the ratio between monomer and oligomer A β, monomer A β, than the less quotient of monomer and oligomer A β, indicates the deterioration of the higher neurological susceptibility of the described disease of generation of described experimenter, higher possibility that described disease exists or the state of an illness.

3. the method for claim 1, wherein step (c) determines the ratio between monomer A β and oligomer A β, monomer A β, than the less quotient of oligomer A β, indicates the deterioration of the higher neurological susceptibility of the described disease of generation of described experimenter, higher possibility that described disease exists or the state of an illness.

4. the method for any one of claim 1, wherein step (c) determines the amount of oligomer A β, the higher amount of oligomer A β, indicates the deterioration of the higher neurological susceptibility of the described disease of generation of described experimenter, higher possibility that described disease exists or the state of an illness.

5. the method for any one of claim 1-4, wherein step (a) and (b) measure at least one in A β x-37, A β x-38, A β x-39, A β x-40, A β x-41 and A β x-42.

6. the method for claim 5, wherein step (a) and (b) at least measure A β x-40.

7. the method for claim 5, wherein step (a) and (b) at least measure A β x-42.

8. the method for claim 5, wherein step (a) and (b) at least measure A β x-40 and A β x-42.

9. the method for any one of claim 1-8, wherein the amount of monomer A β uses one or more antibody to measure, described antibody holds epi-position in conjunction with one or more C-, and described C-holds epi-position exist in monomer A β and do not exist in oligomer A β or be spatially blocked.

10. the method for claim 9, wherein said one or more C-hold antibody to be one or more end-specific antibodies for A β 37, A β 38, A β 39, A β 40, A β 41 or A β 42.

The method of 11. claims 9, one or more C-wherein said hold antibody to comprise the antibody of terminal specificity for A β 40, are optionally antibody 2G3.

The method of 12. claims 9, one or more C-wherein said hold antibody to comprise the antibody of terminal specificity for A β 42, are optionally antibody 21F12.

13. the method for claim 9, wherein said one or more C-hold antibody to comprise terminal specificity for the antibody of A β 40 and the terminal specificity antibody for A β 42.

The method of 14. any one of claim 8-11, wherein said monomer A β is measured by the affine sandwich assay of immunity, and described determination method comprises one or more C-described and holds antibody and the another kind of antibody in conjunction with N-end and/or central epi-position.

The method of 15. claims 14, wherein said another kind of antibody holds epi-position in conjunction with N-, and optionally wherein said antibody is 3D6.

The method of 16. claims 14, wherein said another kind of antibody is in conjunction with central epi-position, and optionally wherein said antibody is 266.

The method of 17. claims 14, one or more C-wherein said hold antibody to be reporter antibody, and described another kind of antibody is capture antibody.

The method of 18. claims 14, one or more C-wherein said hold antibody to be capture antibody, and described another kind of antibody is reporter antibody.

The method of 19. claims 17 or 18, one or more reporter antibody rutheniums wherein said mark, and described capture antibody biotin labeling.

The method of 20. any one of claim 1-19, in step (b), wherein measure the amount of monomer and oligomer A β, comprise with sample described in depolymerization agent process oligomer A β being transformed into monomer A β, and measure the amount of monomer A β in the sample of described depolymerization agent process.

The method of 21. claims 20, wherein said depolymerization agent comprises guanidine hydrochloride, guanidinium isothiocyanate, urea, thiocarbamide, lithium perchlorate and/or potassium iodide.

The method of 22. claims 20, wherein said depolymerization agent comprises non-ionic detergent.

The method of 23. claims 20, wherein said depolymerization agent comprises polyglycol, polyvinylpyrrolidone, polyphenol and/or hexafluoroisopropanol.

The method of 24. claims 20, the amount of monomer A β in the sample of wherein said depolymerization agent process, by measuring for the determination method that the amount measuring monomer A β is identical with step (a).

25. the process of claim 1 wherein that step (a) and (b) are undertaken by quantitative mass spectral art.

26. the process of claim 1 wherein that step (a) and (b) are by kapillary or gel electrophoresis, are then undertaken by quantitative Western blot method.

The method of 27. any one of claim 1-26, wherein said humoral sample is CSF sample.

The method of 28. any one of claim 1-26, wherein said humoral sample is blood sample.

The method of 29. claims 28, wherein said blood sample is plasma sample.

30. the process of claim 1 wherein that step (a) and (b) carry out simultaneously.

31. the process of claim 1 wherein that the sample of step (a) and the second sample of step (b) are the different sample aliquot coming from simple sample.

The method of 32. any one of claim 1-30, wherein said experimenter does not have cognitive impairment, and step (d) assesses the neurological susceptibility that A Zihai Mo's disease occurs described experimenter.

The method of 33. any one of claim 1-30, wherein said experimenter has mild cognitive impairment, and step (d) assesses the neurological susceptibility that A Zihai Mo's disease occurs described experimenter.

The method of 34. any one of claim 1-30, wherein said experimenter has cognitive impairment, and step (d) comprise use step (c) comparison and other symptoms of described experimenter's illness and/or the combination of sign to provide the diagnosis of A Zihai Mo's disease.

The method of 35. any one of claim 1-30, wherein said experimenter had been diagnosed as before carrying out described method suffers from A Zihai Mo's disease, and step (d) provides the instruction of described disease stage.

The method of 36. any one of claim 1-30, wherein said experimenter is just accepting treatment or the prevention of A Zihai Mo's disease, and step (d) provides described experimenter to the instruction of the response for the treatment of.

The method of 37. claims 36, wherein said method is carried out at set intervals, and the instruction providing the response to treatment more over time of step (c).

The method of 38. claims 36, wherein said experimenter is just with the Immuno Suppressive Therapy for A β.

The method of 39. claims 36, wherein said experimenter is just with hundred flat pearl monoclonal antibody treatments.

The method of 40. claims 39, before it is also included in and carries out step (a) and (b), with the anti-idiotype for hundred flat pearl monoclonal antibodies, optionally with the described sample of JH11.22G2 process.

The method of 41. any one of claim 1-40, it also comprises the amount measuring Tau or P-Tau in described sample, wherein Tau or P-Tau is relative to the increase of control value, provides the neurological susceptibility of generation A Zihai Mo's disease of described experimenter, the existence of A Zihai Mo's disease or the further instruction that sb.'s illness took a turn for the worse.

The method of 42. claims 2, wherein said experimenter enters clinical testing to test the candidate for the treatment of A Zihai Mo's disease or the medicine of prevention, if wherein described monomer A β than the quotient of monomer and oligomer A β lower than threshold value, then described experimenter is comprised in described clinical testing, if described experimenter is higher than described threshold value, then described experimenter is excluded from described clinical testing.

The method of 43. any one of claim 1-42, it also comprises the nursing supplier described diagnosis, prognosis or monitoring being informed described experimenter or described experimenter.

The method of 44. aforementioned any one of claim, wherein the step (c) of at least described method performs in a computer.

The method of 45. claims 44, wherein said computing machine receives the signal relevant with the amount of monomer A β and the amount of monomer and oligomer A β, described signal is transformed into quantitative amount, more described amount quantitatively, and provide and treat relevant output with the recommendation of the comparison of described amount, described amount, the situation of described experimenter or described experimenter.

Determine that, to which the snibject's medicine in colony with the method for the prevention or treatment of carrying out A Zihai Mo's disease, described method comprises each experimenter in described colony for 46. 1 kinds:

A. the amount of monomer A β in humoral sample is measured;

B. the amount of monomer and oligomer A β in the second sample of body fluid is measured; And

C. the amount of the amount of monomer A β and monomer and oligomer A β is compared,

Wherein on basis of the comparison, experimenter in described colony accepts or does not accept medicine to treat A Zihai Mo's disease or to carry out the prevention of A Zihai Mo's disease.

The method of 47. claims 46, wherein said comparison determines monomer A β and the ratio between monomer and oligomer A β, and wherein monomer A β accepts described medicine than the quotient of monomer and oligomer A β lower than the experimenter of threshold value.

The method of 48. claims 46, it carries out according to any one of claim 1-44.

49. 1 kinds of methods determining which kind of therapeutic scheme of experimenter given in colony, described method comprises each experimenter in described colony:

A. the amount of monomer A β in humoral sample is measured;

First sub-group of wherein said experimenter is treated with the first therapeutic scheme, second sub-group of described experimenter is treated with the second therapeutic scheme, wherein between the experimenter and the experimenter of the second sub-group of described first sub-group, monomer A β is significantly more different than the ratio of monomer and oligomer A β.

The method of 50. claims 49, wherein said first therapeutic scheme comprises the medicine of prevention for A Zihai Mo's disease or treatment, described second therapeutic scheme does not comprise described medicine, and the experimenter of described first sub-group has the ratio of lower monomer A β than monomer and oligomer A β compared with the experimenter of described second sub-group.

The method of 51. claims 49 or 50, wherein monomer A β than the quotient of monomer and oligomer A β in the experimenter of described first sub-group lower than threshold value, and lower than threshold value in the experimenter of described second sub-group.

The method of 52. any one of claim 49-51, it carries out according to any one of claim 1-44.

53. 1 kinds are carried out the method for difference treatment to the experimenter in colony, described method comprises first sub-group for the treatment of described experimenter with the first therapeutic scheme, and second sub-group of described experimenter is treated with the second therapeutic scheme, the experimenter in the experimenter in wherein said first sub-group and described second sub-group has the average ratio of significantly different monomer A β than monomer and oligomer A β.

The method of 54. claims 53, experimenter's prevention of wherein said first sub-group or the drug therapy for the treatment of A Zihai Mo's disease, the experimenter of described second sub-group need not described drug therapy, and compared with the experimenter in described second sub-group, monomer A β is than the ratio of monomer and oligomer A β, obviously lower in the experimenter of described first sub-group.

55. the method for claim 53 or 54, wherein monomer A β carries out measuring and comparing according to any one of claim 1-44 than the amount of monomer and oligomer A β.

56. determine a method of which experimenter in colony being called up in clinical testing, described method comprises each experimenter in described colony:

A. the amount of monomer A β in humoral sample is measured;

Wherein on basis of the comparison, the experimenter in described colony called up or do not call up in described clinical testing.

The method of 57. claims 56, wherein said comparison determines monomer A β and the ratio between monomer and oligomer A β, and the experimenter that wherein monomer A β falls within threshold value than the quotient of monomer and oligomer A β is called up in described clinical testing.

58. 1 kinds of diagnostic kits, it comprises:

At least one terminal specificity holds antibody for the C-of A β 37, A β 38, A β 39, A β 40, A β 41 or A β 42;

In conjunction with the N-end of A β and/or the antibody of central epi-position; And

Oligomer A β is transformed into the depolymerization agent of monomer A β.

The diagnostic kit of 59. claims 58, wherein said C-holds antibody terminal specificity for A β 40 or A β 42.

60. the diagnostic kit of claim 58, it comprises terminal specificity and holds antibody and terminal specificity to hold antibody for the C-of A β 42 for the C-of A β 40.

61. 1 kinds of screenings have the method for the medicament of the activity of antagonism A Zihai Mo's disease, and described method comprises:

The transgenic rodents model of A Zihai Mo's disease is contacted with described medicament;

The amount of monomer A β and the amount of monomer and oligomer A β in the body fluid of the transgenic rodents relatively contacted with described medicament; And

Use and describedly relatively determine whether described medicament has the activity that can be used for treating A Zihai Mo's disease.

62. 1 kinds of methods analyzing A β, described method comprises:

A. measure the amount coming from A β in the humoral sample of experimenter, depolymerization agent process do not used by wherein said sample;

B. the amount coming from A β in the humoral sample of described experimenter is measured, the process of wherein said sample depolymerization agent; And

C. the amount measured in step (a) and (b) is compared.

63. the method for claim 62, wherein said step (a) and the measurement in (b) use terminal specificity to carry out for the C-end antibody of A β.

The method of 64. claims 62, wherein said compare the amount in step (a) that determines and the amount in step (b) ratio or step (a) and step (b) in amount between difference.

The method of 65. claims 62, it also comprises:

D. described ratio or difference are used for the diagnosis of A Zihai Mo's disease or its neurological susceptibility in described experimenter, prognosis or monitoring, amount in step (a), than the higher difference between the amount in the lower quotient of the amount in step (b) or step (b) and step (a), indicates the deterioration of the higher neurological susceptibility of the described disease of generation of described experimenter, higher possibility that described disease exists or the state of an illness.

The method of 66. any one of claim 62-65, wherein step (a) and (b) measure at least one in A β x-37, A β x-38, A β x-39, A β x-40, A β x-41 and A β x-42.

The method of 67. any one of claim 62-65, wherein step (a) and (b) at least measure A β x-40.

The method of 68. any one of claim 62-65, wherein step (a) and (b) at least measure A β x-42.

The method of 69. any one of claim 62-65, wherein step (a) and (b) at least measure A β x-40 and A β x-42.

The method of 70. any one of claim 62-65, wherein the amount of A β uses one or more terminal specificity to carry out for the C-end antibody of A β 37, A β 38, A β 39, A β 40, A β 41 or A β 42.

71. the method for claim 70, wherein said one or more C-hold antibody to comprise terminal specificity for the antibody of A β 40 and the terminal specificity antibody for A β 42.

The method of 72. claims 70 or 71, wherein A β is measured by the affine sandwich assay of immunity, and described determination method comprises one or more C-described and holds antibody and the another kind of antibody in conjunction with N-end and/or central epi-position.

The method of 73. any one of claim 62-72, wherein said depolymerization agent comprises guanidine hydrochloride, guanidinium isothiocyanate, urea, thiocarbamide, lithium perchlorate and/or potassium iodide, non-ionic detergent, polyglycol, polyvinylpyrrolidone, polyphenol and/or hexafluoroisopropanol.

The method of 74. any one of claim 62-73, wherein step (a) and (b) use identical determination method to measure the amount of A β.

The method of 75. any one of claim 62-74, wherein said humoral sample is CSF sample or blood sample.

The method of 76. any one of claim 62-75, wherein said experimenter does not have cognitive impairment, and step (d) assesses the neurological susceptibility that A Zihai Mo's disease occurs described experimenter.

The method of 77. any one of claim 65-75, wherein said experimenter has mild cognitive impairment, and step (d) assesses the neurological susceptibility that A Zihai Mo's disease occurs described experimenter.

The method of 78. any one of claim 65-75, wherein said experimenter has cognitive impairment, and step (d) comprise use step (c) comparison and other symptoms of described experimenter's illness and/or the combination of sign to provide the diagnosis of A Zihai Mo's disease.

The method of 79. any one of claim 65-75, wherein said experimenter had been diagnosed as before carrying out described method suffers from A Zihai Mo's disease, and step (d) provides the instruction of described disease stage.

The method of 80. any one of claim 65-75, wherein said experimenter is just accepting treatment or the prevention of A Zihai Mo's disease, and step (d) provides described experimenter to the instruction of the response for the treatment of.

81. the method for claim 80, wherein said method is carried out at set intervals, and the instruction providing the response to treatment more over time in step (c).

The method of 82. claims 80 or 81, wherein said experimenter is just with the Immuno Suppressive Therapy for A β.

The method of 83. claims 82, wherein said experimenter is just with hundred flat pearl monoclonal antibody treatments.

The method of 84. claims 83, before it is also included in and carries out step (a) and (b), with the anti-idiotype for hundred flat pearl monoclonal antibodies, optionally with the described sample of JH11.22G2 process.

The method of 85. any one of claim 62-84, it also comprises the amount measuring Tau or P-Tau in described sample, wherein Tau or P-Tau is relative to the increase of control value, provides the neurological susceptibility of generation A Zihai Mo's disease of described experimenter, the existence of A Zihai Mo's disease or the further instruction that sb.'s illness took a turn for the worse.

The method of 86. any one of claim 62-85, it also comprises the nursing supplier described diagnosis, prognosis or monitoring being informed described experimenter or described experimenter.

The method of 87. claims 62, its experimenter in colony carries out, first sub-group of wherein said experimenter is treated with the first therapeutic scheme, second sub-group of described experimenter is treated with the second therapeutic scheme, and compared with the experimenter of described second sub-group, the ratio of amount ratio amount of the A β of measurement in step (b) of the A β measured in step (a) is obviously lower in the experimenter of described first sub-group.

88. the method for claim 87, wherein said first therapeutic scheme comprises the medicine of prevention for A Zihai Mo's disease or treatment, and described second therapeutic scheme does not comprise described medicine.

The method of 89. claims 87 or 88, the ratio of amount ratio amount of the A β of measurement in step (b) of the A β wherein measured in step (a), lower than threshold value in the experimenter of described first sub-group, and higher than described threshold value in the experimenter of described second sub-group.